Laboratory Manual 

of 

General Agricultural 
Bacteriology 

Hastings and Wright 





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A Laboratory Manual 

of 

General 

Agricultural 

Bacteriology 



v J 

E. G. HASTINGS 

Prosessor of Agricultural Bacteriology 

University of Wisconsin 

and 

W. H. WRIGHT 

Associate Professor of Agricultural Bacteriology 

University of Wisconsin 










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1*124 


Copyrighted 1924 

by 

Grimm Book Bindery 
Madison, Wis. 



© Cl A 80 0900 

♦ 


The Print Shoo, Printers 



Preface to the First Edition 


With the present day tendency in our agricultural colleges to crowd much 
into the limitations of the curriculum, the problem of what to give and what 
not to give in a general course becomes a perplexing one. 

More than ten years of experience teaching agricultural, home economics, 
science and engineering students have contributed to the development of the 
present edition of the Laboratory Manual of General Agricultural Bacteriology. 
Many of the changes that have been made from time to time have come as 
the result of class room experience and the need for more economical utiliza¬ 
tion of the time of both instructor and student. 

No attempt is made to cover completely and in detail all phases of mor¬ 
phology, cultural characters and physiology of. bacteria. Enough of each 
of these is included in connection with practical exercises to give the student 
a general conception of the relation of bacteria to agricultural processes. 
For those who wish more detailed information the advanced courses are 
available; while the students going into other lines of agriculture taking it 
as a required course find themselves broadened by a general knowledge of 
the relation of bacteria to practical things. 

The exercises as outlined have been planned for local use. No effort has 
been made to arrange them for all kinds of conditions. On this account their 
use elsewhere may require some slight modifications. The number and 
variety of the exercises offer considerable latitude in the choice of work for 
a course. This is desirable where the type of the students in the course 
changes from semester to semester. 


THE AUTHORS. 




> 


Preface to the Second Edition 


The reception of the first edition of the Laboratory Manual of General 
Agricultural Bacteriology has been very satisfactory. The issuing of a second 
edition, with such changes as may be considered necessary, is primarily for 
the needs of the authors’ classes. 

The plan of the book has not been materially changed, but more detail 
has been introduced into several of the exercises. These changes are con¬ 
sidered desirable because the laboratory time of the student has been in¬ 
creased considerably. 

It is hoped that others who have been using the manual will find the 
changes equally acceptable and adapted to their needs. 

University of Wisconsin THE AUTHORS. 

September, 1924. 




PART I 


GENERAL DIRECTIONS 

LABORATORY REGULATIONS 

1. Desks and Lockers. Each student is assigned a desk and given an 
individual locker in this desk. The student will be held responsible for the 
condition of the desk at the end of each meeting of his laboratory section. 

2. Apparatus. The apparatus needed in the laboratory work will be 
found in the locker. The apparatus should be checked with the list placed 
on the blackboard by the instructor. Missing articles should be obtained from 
the storeroom before the locker is accepted. Any damaged article should be 
exchanged at the storeroom. 

3. Supplies. The materials needed for the work of each laboratory 
period will be placed on the desk before the section assembles. Additional 
supplies, which may be needed from time to time, may be secured at the 
storeroom. On one of the white cards to be found in the metal box on the 
desk, place your name, locker number, and the supplies you wish. Present 
the card thus made out to the storeroom. 

4. Return of Apparatus. On one of the blue cards to be found in the 
metal box on the desk, place your name, locker number, and list of the 
apparatus you are to return. Present the card, together with the apparatus, 
at the storeroom. 

All glassware returned to the storeroom must be clean and dry. Ap¬ 
paratus is to be returned at such times and in such amounts as may be an¬ 
nounced. 

5. Waste Material. All waste paper, cotton plugs, matches, and broken 
glassware must be placed in the central compartment of the metal box to 
be found on each desk. 

6. Washing Apparatus. All dirty apparatus is to be washed at the large 
wall sinks. The small sinks are for staining purposes only. 

7. Arrangement of Desks. At the end of each laboratory period each 
student must place his desk in order: 

(a) Return all bottles to tray. 

( b ) Place all waste material in the box. 

( c) Return the stencil to its place. 

(d) Wipe off the entire desk top with a damp sponge. 

(e) Place the stool under the desk. 

8. Microscopes. Whenever possible each student is assigned an in¬ 
dividual microscope. After assignment, the one using the microscope will 
be charged for any injury or neglect happening to the instrument during 
the regular scheduled laboratory periods at which he works. The microscope 
should be examined as soon as it is removed from the locker to see that it is in 
working order. The microscope must be returned to the locker at the end of 
the laboratory period. 











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2 


9. Regulation Laboratory Periods. It is expected that the work as¬ 
signed each day will be done in the regular laboratory period of two hours. 
The work is so arranged that this is possible for the average student. Students 
absent or having to do work outside of the regular laboratory hours for any 
valid reason, may arrange with the instructor to do such work at a time when 
there is not a regular class in the laboratory. 

10. Laboratory Notes. All laboratory notes and drawings are to be made 
on the prescribed paper and in the following manner. 

(a) Each exercise number and title must be entered as in the 
manual. 

( b ) All experiments are to be written up under the headings of 

I. Object. 

II. Method. 

III. Results. 

IV. Conclusions. 

( c ) In presenting results, the data should be tabulated whenever 
possible. 

The following conditions should be observed when recording results: 

1. In writing up experiments use the indicative mode, passive voice, 
third person. 

2. Do not use abbreviations. 

3 V Put down your own conclusions as well as answers to questions. 

4. Include important details. 

5. All exercises asked for must be in by Saturday noon of each week. 

(d) Drawings should always be located on the left hand margin 
of the page. The outline stencil should be used for drawings 
of test-tube cultures and of microscopic preparations. All 
drawings should be properly labeled underneath and the 
description should be to the right of the same page. 

( e) The pages of all the notes are to be numbered consecutively 
from the beginning of the course. 

(/) Notes are to be placed on only one side of each sheet. 

(g) The notes for each week’s work should be placed in the 
regulation envelope when turned in. They will be graded 
and returned the succeeding week. 


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PART II. 


STUDY AND CULTIVATION OF BACTERIA 
Exercise 1. Construction and Operation of the Microscope 
MATERIALS: 

A compound microscope completely equipped 
A test object mounted on a slide 

1. The type of compound microscope used in bacteriological work repre¬ 
sents the highest development in the fields of optics and mechanics. The 
mechanical parts are easily injured; the lenses, especially those giving great 
magnifications, are delicate and expensive. The instrument should be han¬ 
dled with care. 

2. Notice that the compound microscope (Fig. 1) is provided with a foot, 
A; arm, B; stage, C; tube, D; nose piece, E; objectives, F and G; Abbe condenser, 
H; iris diaphragm, I; ocular, J; mirror, K; coarse adjustment, L; and fine adjust¬ 
ment, M. 



FIG. 1-CONSTRUCTION OF THE COMPOUND MICROSCOPE 


























































































4 



FIG. 2— EFFECT OF DIVERGENT RAYS 
ENTERING THE CONDENSER. 

The rays come to a focus below the object and 
the illumination is reduced. 



The rays are brought to a focus in the plane 
where the object lies. 


3. The Abbe condenser, H, receives the light falling on an area of the mir¬ 
ror equal in size to the lower lens of the condenser, and concentrates it on a 
much smaller area. (See Figs. 2 and 3). This increases the illumination and 
brilliance of the object, a result which is very important in the operation of 
high power objectives. 

4. The iris diaphragm, I, provides a means by which the amount of light 
entering the front lens of the objective may be regulated. It is especially of 
value when using the low power objectives because with them the definition 
and detail of the object is brought out more clearly when the light is not too 
intense. 


5. The oil-immersion objective gives the highest magnification. The front 
lens is very small and hence admits but little light. In order that the objects 



FIG. 4-SHOWING THE PRINCIPLE OF THE IMMERSION OBJECTIVE 



























5 

viewed with it may be seen distinctly, the light must be concentrated by 
means of the condenser, and be conserved by means of a liquid, e. g. oil of 
cedar, having the same index of refraction as glass. The oil fills the space 
between the object and the objective. In this way the loss of light is prevented 
as shown in Fig. 4. 

6. The ocular or eyepiece, J, into which one looks when using the micro¬ 
scope, forms an image of the image formed by the objective; hence the term» 
compound microscope. Oculars of varying powers of magnification can be 
used, thereby enabling one to obtain any desired magnification, the practical 
working limit being 1200 to 1500 diameters. 

7. The mirror, K, is used to reflect the light into the condenser. One side is 
plane or flat for use with daylight, and the other side is concave for use with 
artificial light. In either case parallel rays are reflected into the condenser. 
(See Figs. 2 and 3.) 

8. Place the microscope in a vertical position on the desk with the coarse 
adjustment next you and near enough that you can look into the ocular easily. 

9. Lower the tube with the coarse adjustment until the front lens of the 
objective is within 6 m. m. of the stage. See that the iris diaphragm below 
the condenser is open. 

10. Remove the ocular. Swing the low power objective into position. 
While looking into the tube, adjust the mirror so that an image of the electric 
bulb is seen in the center of the lighted area, which is known as the field. 
Replace the ocular. The field should be uniformly bright. If it is not, alter 
the position of the mirror until it is uniformly lighted. 

(a) Turn the mirror slightly to the right. What is the effect? 

Turn the mirror slightly forward. What is the effect? 

( b ) Close the diaphragm. What is the effect? 

(c) Lower the condenser slowly while looking into the ocular. 

What is the effect? 

11. Whenever starting to use the microscope, examine it to determine that 
the iris diaphragm is open, the condenser is up as high as it will go and the 
concave side of the mirror is up. Place the instrument near the edge of the 
desk facing directly in. Adjust the light as described in paragraph 10. Do 
not move the microscope for the purpose of obtaining even illumination of the 
field. Use the mirror for this purpose. 

12. Examine a permanent, mounted preparation of the leg and wing of the * 
common house fly. 

13. In focusing the microscope upon any object to be viewed, the proper 
objective and ocular being in place, the tube is moved down by the coarse 
adjustment, L. until the objective is near the object, but not touching it. 
Looking into the ocular, the motion is reversed. When the image begins to 
show, the movement must become slow. 

14. With the lowest power a good focus can usually be obtained with the 
coarse adjustment. However, with the high powers the fine adjustment, M, 
must be used in order that the tube may be moved slowly. The fine adjust¬ 
ment works on a fine thread so that a complete revolution of the screw moves 
the tube very little. Care should be taken not to turn the screw completely 
down until it is “jammed” tight or so far upward that it is off the thread. 



6 


15. Place in the notes the following: 

(а) Make of the microscope. 

(б) The number of the ocular. 

(c) The numbers of the objectives. 

( d ) The magnifications obtained with the ocular and each of the 
objectives on the microscope. See table below. 

(e) By what characteristic can one recognize at a glance the 
lowest power objective? the oil immersion objective? 

(f) Which way must the coarse adjustment be turned to raise the 
tube? 

( g ) Which way must the fine adjustment be turned to raise the 
tube? 

( h) The condenser can be raised and lowered in two ways. What 
are they? 

(i) The image of the object viewed is an inverted one. What 
effect does this have on the direction the slide must be 
moved in order to move an object seen in the field from left to 
right? 

(/) The values given in the table of magnification refer to the 
apparent increase in length of a line as viewed under the 
microscope—linear magnification. From these values how 
would the magnification of an area be obtained? 

16. The first five minutes of the next period will be devoted to a written 
quiz on the names applied to the various parts of the microscope and the 
purpose of each part. 


TABLE OF MAGNIFICATIONS 
(tube length of 160 m. m.) 


Objectives 

Oculars 


4x 

5x 

8x 

lOx 


(2 in.) 

(2 in.) 

(1 in.) 

(1 in.) 

16 m. m. (f in.) — 

40 

50 

80 

100 

4 m. m. (£ in.)- 

176 

220 

352 

440 

1.8 m. m. (tV in.). 

380 

475 

760 

950 
















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7 


Exercise 2. Examination of Objects Under tlie Microscope 
MATERIALS: 


3 Plain glass slides 
2 Cover glasses 
Clean sand 

Copper chloride crystals 
Cotton fibers 
Wool fibers 


1. On a clean slide place near the center a few grains of sand and a few copper 
chloride crystals. 

2. Adjust the microscope. 

3. Place the slide on the stage of the microscope with the objects to be 
examined over the opening. See that the clamps hold the slide securely. 

4. Swing the low power (16 m. m.) objective into position and lower the 
tube until the tip of the objective is within 4 m. m. of the object on the 
slide. 


5. While looking into the microscope, slowly raise the tube by turning the 
coarse adjustment toward you until the objects come into view. It is well to 
move the slide a very little while focusing in order that the objects may more 
easily be detected. Do not mistake shadows of your eyelashes, or dust and 
lint particles on the lenses of the eyepiece for the objects to be examined. 
Turn the eyepiece to see if any such particles move. If so, they are on the 
eyepiece. If the lenses are very dirty, clean them with lens paper. If this 
does not remove the dirt, ask the instructor to clean them for you. 

6. Try changing the amount of light by opening and closing the diaphragm. 
Draw the objects as seen with the 16 m.m. objective, remembering the regula¬ 
tions for drawings. (See page 2, paragraph 10.) 

7. Place a large drop of water on a slide. In the water place a few wool 
fibers. Clean a cover glass. Hold it inclined so that the lower edge touches 
the slide and the edge of the drop. Lower it carefully in order that the space 
beneath the cover glass will be completely filled with water and not with a 
mixture of water and air. Remove excess of water with a blotter by blotting 
gently as when blotting ink. Examine the fibers with the 16 m.m. objective. 
Draw and describe them as seen with this objective. 

8. Examine the fibers with the 4 m. m. objective. Draw them as seen with 
this objective. 

9. Examine some cotton fibers in the same manner. Draw them as seen 
with the 4 m. m. objective. 


10. Examine some threads, warp and woof, from your suit. Is it all wool? 


11 . 


(a) How do the numbers of the objectives correspond to their 
distances from the objects when in focus? 

( b ) With what intensity of light can the details of the wool be 
most easily distinguished? 

(c) What is the structure of the wool fiber as shown by its appear¬ 
ance under the microscope? 

( d ) What is the structure of the cotton fiber as shown by its 
appearance under the microscope? 


I 


8 


Exercise 3. Examination of Stained Dead Bacteria 
MATERIALS: 

Prepared slides of the principal morphological types of stained, 
dead bacteria. 

1. Adjust the microscope as before. See that the diaphragm is completely 
open and the lighting is even. 

2. Place the slide of stained or dyed specimens of dead bacteria upon the 
stage of the microscope. 

3. Examine preparation A with the 16 m. m. objective. 

(a) Is there any detail or anything definite to be seen? 

(b) Draw what can be seen. 



FIG. 5-FORM AND ARRANGEMENT OF THE LOWER BACTERIA 

A, spheres; B, rods; C, spirals 


4. Examine the same preparation with the 4 m. m. objective. 

(a) What can be seen? 

( b ) Draw a few of the objects seen. 

Make the drawings large enough to show the exact shape of 
the objects and much larger in proportion to the size of the 
stencil than the objects bear to the microscopic field. 

5. Raise the tube until the 4 m. m. objective is not less than 4 m. m. above 
the object. Place a small drop of immersion liquid on the center of the cover 
glass. Revolve the oil immersion objective into place. Carefully lower it, 
watching from one side, until the lens of the objective is immersed in the oil 
and almost touches the object. 





























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9 


6. While looking into the ocular, and while moving the slide slowly back 
and forth, turn the fine adjustment so that the tube moves upward. Turn 
slowly until the image appears sharp and clear. 

7. Draw and describe preparation A as seen with the oil immersion object¬ 
ive. It is not necessary to draw all of the organisms seen in a field. The 
drawings should be large enough to show the exact shape of the organisms in 
outline. 

8. Examine preparation B with the oil immersion objective. Draw a field 
from the prepapration. 

9. Examine preparation C with oil immersion objective. Draw a field 
from the preparation. 

10. According to shape, name the bacteria in preparations A. B. and C. 
(See Fig. 5.) 

11. Draw and describe the organisms in preparations D. showing spores 
and in E. showing flagella. 

12. Knowing the diameter of the field of the oil immersion objective, 
(see paragraph 14), estimate the size in millimeters of the organisms in pre¬ 
parations A and B. 

13. The micron (written y) is the standard of measurement for bacteria. 
1 micron—0.001 m. m. 

(a) Give the estimated size of the organisms in microns. Show 
the calculations in each case. 

14. The diameters of the real fields of the objectives most commonly used 
are given in the following table: 


DIAMETER OF REAL FIELDS OF OBJECTIVES 


Objectives. 

Diameter of Field 

Using lOx (1 inch) Eyepiece 
and a tube length of 160 m. m. 

16 m. m. (f in.) 

1.55 m. m. 

4 m. m. G in.) 

0.31 m. m. 

1.8 m. m. (^ in.) 

0.16 m. m. 


Note.—As soon as the work with the oil-immersion objective is completed, 
it should be wiped off with lens paper. 










10 


Exercise 4. Microscopic Study of Living Bacteria 

MATERIALS: 

Carmine powder in water 
Hay infusion 
Whole milk 

Pure cultures of bacteria in meat broth 
Hanging-drop slide 
Cover glasses 
Vaseline 

1. Clean a hanging drop slide. Place a ring of vaseline around the edge of 
the depression. Clean a cover glass and lay it on a clean piece of paper. 

2. With the wire loop place a small drop, about the size of a pin head, of a 
suspension of carmine in water on the center of the cover glass. 

Note. —Whenever the wire loop or needle is used, it must be sterilized 
immediately before and after using, in the manner shown by the instructor. 

3. Invert the slide and carefully lower it until the cover glass is touching 
the ring of vaseline on all sides. 

4. Quickly turn the slide right side up so that the drop is suspended (hangs) 



FIG. 6-CORRECT HANGING DROP 


XX 


FIG. 7-WRONG OR “FALLEN” HANGING DROP 

over the depression in the slide. (See Fig. 6.) Carefully press down on the 
edge of the cover glass so that the vaseline makes a seal. Be careful not to 
cause the drop to fall or touch the bottom of the depression. (See Fig. 7.) 
In case this happens clean the slide and cover glass and repeat. 

5. Examine the drop with the 16 m. m. objective. Shut off most of the 
ight in order that the drop may be seen distinctly. Find the edge of the drop. 

6. Raise the 16 m. m. objective a little and revolve the 4 m. m. objective 
into place. Regulate the light (usually by admitting more of it) until the field 
is grayish. 

7. Looking at the objective sideways, carefully lower it until it nearly 
touches the cover glass. 

8. Looking into the eyepiece, focus upward very slowly with the fine 
adjustment until the particles of dye are plainly visible. Try regulating the 
diaphragm until the best light is secured. 

9. Examine the smaller carmine particles and groups of particles. 

(a) What is their color and shape? 

( b) Do they move? 

(c) Is the movement vibratory, or do the particles move from one 
place to another in the preparation? 

The vibratory form is called pedesis or brownian movement. 

10. Examine some diluted milk (1 part whole milk to 2 parts water) in a 
like manner. 













11 

11. Examine a dark-field preparation of carmine particles. See the instruc¬ 
tor’s demonstration. 

12. Great numbers of bacteria of different kinds will develop in a jar 
containing water and grass, hay or leaves, etc. The soluble substances in these 
diffuse into the water and serve as food for bacteria and other micro-organ¬ 
isms. The micro-organisms that were on the hay and leaves, in the water or 
adhering to the jar or those that are introduced with dust from the air develop 
with amazing rapidity. This development will take place at temperatures 
varying from 15 degrees G. to 40 degrees C. and is an example of what is 
taking place in nature wherever food and moisture permit the growth of 
bacteria. 

Prepare a hanging drop from the infusion on the desk and locate the drop in 
the same way as before. 

13. After locating the edge of the drop with the 16 m. m. objective, examine 
it with the 4 m. m. objective. Focus upward very slowly with the fine adjust¬ 
ment until the objects are in view. The organisms in the shoal water are the 
most easily studied. The large, round, oval and oblong objects that seem to 
move about so rapidly are not bacteria, but animals. Animals like these are 
known as protoza. The small, round, rod, or spiral-shaped individuals 
present are the bacteria. 

14. Draw and describe afield as seen in the hanging-drop preparation. 

(a) Are there a number of kinds of bacteria in the infusion? Give 
reasons for your answer. 

(b) Can any of them move? Indicate the path of the movement 
of individual organisms by dotted lines and arrows. 

(c) What seems to be the nature of their movement? Notice the 
apparent vibration of some of them that are not moving from 
place to place. 

15. Examine in hanging drop each of the pure cultures of bacteria furnished; 
draw and describe each. 

16. From the observations of the cultures, classify each one according to 
shape. (See Fig. 5). 

(a) Unstained, living bacteria have been examined as well as 
stained, dead bacteria. Which one of these preparations is 
the easier to study? Why? 

( b ) Can flagella be seen on the bacteria in any of the preparations 
Give the reason for your answer. 

(c) Can dead bacteria be detected by observation of a hanging 
drop? Give reasons for your answer. 


# 





12 


Exercise 5. Microscopic Study of Living Yeast and Molds 
MATERIALS: 

Culture of yeast on dextrose agar 
Cultures of molds on potato agar 

1. Place three or four drops of the yeast suspension upon a plain slide. 
Cover with a clean cover glass. The cover glass should float on the liquid 
which should be sufficient in amount to extend under the cover glass, but 
should not cover the upper surface so that it comes in contact with the 
objective. 

2. Examine the preparation with a 4 m. m. objective. Draw and describe 
the organisms. 

3. Examine the pure cultures of molds in the Petri dishes. Describe their 
naked eye appearance. 

4. Place the dish on the stage of the microscope with the cover removed. 
Examine the patches, preferably the edge, with the 16 m. m. objective. 
Notice the color, form, and size of the patch of mold. Notice the filaments 
which grow on the jelly-like food substance and the erect ones carrying the 
spores. 

5. Make a more detailed examination of the filaments and spores using the 
4 m. m. objective. 

6. Make a large detailed drawing of each of the molds furnished. In each 
case show the following: 

(a) Mycelium and filament structure. 

(b) Fertile hyphae. 

(c) Sporangia and spores. 

(tf) Label all of the parts and give the/genus of each mold studied. 

7. (a) How do molds differ from bacteria. 

(b) How do they differ from yeasts? 

(c) How do the molds reproduce? 

( d ) How yeasts reproduce? 

( e ) How do yeasts compare in size with bacteria? 



13 


Exercise 6. Making Stained Preparations of Bacteria 

MATERIALS: 

Hay infusion 

Cultures of bacteria on agar 
Clean slides 
Clean cover glasses 
Canada balsam 
Tooth picks 

1. Examine the infusion with the naked eye. Note whether it has changed 
since it was used in Exercise 4. Look up the meaning of zoogloeae in Appen¬ 
dix B. 

2. Wet the tip of one finger, rub it on a cake of Bon Ami, and with the same 
finger spread the paste thus formed over the surface of the slide. Allow the 
paste to dry, and wipe it off with a clean dry cloth or paper towel. If the slide 
is clean, a drop of water placed on it will spread in an even film over the glass. 
Grease causes water to collect in tiny drops. 

Place a small drop of the infusion in the center of the clean slide. Spread 
the drop over an area about the size of a dime. (See Fig. 8.) The wire loop 
can best be used for this purpose. 



3. Permit the spread on the slide to dry. Do not heat the slide to hasten 
the drying. 

4. Pass the slide, film side up, quickly three times through the Bunsen flame. 
(See Fig. 9.) The heat kills the organisms and causes them to adhere to the 
glass throughout subsequent operations. It is termed “fixing” the prepara¬ 
tion. 












14 



FIG. 9—FIXING THE ORGANISMS TO THE SLIDE 

5. Lay the slide on the sink cover for staining and pour enough methylene 
blue solution on it to cover the spread as is shown in Fig. 10. 

Permit the stain to act three minutes, and then wash the preparation" with 
tap water. 



6. Dry the preparation between blotters. 

7. Label the slide, being careful to place the label at one end on the same 
side as the spread. 

Note.—This kind of a preparation answers very well for all temporary 
work where preparations are not desired for permanent use. 

In order to make the preparations more nearly permanent, the smear or 
spread may be covered with a very thin piece of glass, a cover glass, 
which may be cemented to the slide with Canada balsam. The preparation 
should be dry before the Canada balsam is placed on it. If immersion oil has 
been used on the preparation, it should be removed by flooding the spread 












































• ■ 

■ 








15 

twice with xylol, this being allowed to act a few moments each time before it 
is drained off, after which treatment, the slide is allowed to dry. The Canada 
balsam, dissolved in xylol, should be of such a consistency that a drop one- 
eighth of an inch in diameter will spread under the weight of the cover glass 
so as to fill the space beneath the cover glass completely. The cover glass 
should not be dropped on the Canada balsam, but should be held in an inclined 
position and very slowly lowered on the drop. The preparation should be 
placed in a horizontal position for several days until the balsam has hardened. 

8. Remove some of the tartar from the teeth with a tooth pick and spread 
m a thin, even film without water. Fix and stain three minutes with methy¬ 
lene blue. 

9. Prepare a spread of some old milk which is not curdled. Be sure the 
spread is thin. Do not dilute with water. Dry and immerse in xylol three to 
five minutes to remove the fat. Wash in alcohol to remove the xylol. Wash 
in w r ater. Stain three to five minutes with methylene blue. 

10. From each of the pure cultures of bacteria furnished make a preparation 
in the following manner: 

\\ ith the wire loop place a small drop of tap water on a clean slide. Trans¬ 
fer a minute portion of the bacterial growth on the solid nutrient agar to the 
drop of water by touching the growth with the tip of the wire needle. Mix the 
bacteria with the w r ater, w r hich should then be spread in an even film over an 
area as large as a dime. The turbidity produced in the drop of water by mix¬ 
ing the bacteria in it should be evident to the eye. Allow the preparation to 
dry. Fix it and stain with the dye indicated by the instructor. 

11. Examine the preparations in order, as was done in Exercise 3, using the 
oil immersion (1.8 m. m.) objective only. Draw and describe one good field 
from each preparation. 

(a) What can you see in the infusion? 

(b) How r can you tell bacteria from debris? 

(c) Can you tell the protozoa from the bacteria? 

( d ) How' many different forms of bacteria are visible? Compare 
them with Fig. 5. 

C e ) What can be seen in the tartar? 

(0 Do spheres, rods or spirals predominate? 

(g) Are there many different forms of bacteria in milk? 

(h) What is the amorphous blue material in the stained milk 
spread? 

( i ) What morphological types of bacteria were found in the pure 
cultures? 

(j) What is the value of anilin dyes in the study of bacteria? 


16 


Exercise 7. Sterilization 

MATERIALS: 

Beef broth 
Skimmed milk 
Eight empty test tubes 
Two sterile test tubes 
Cotton batting 

Note. —Where the test tubes contain cultures and experimental material, 
they are handled in tumblers, and to avoid breaking the tubes a cushion of 
cotton is placed in the bottom of each tumbler. 

1. Prepare five tubes of milk and five tubes of beef broth according to the 
following outline, making plugs as shown by the instructor. 


No. 

Material 

Kind of tube 

Treatment 

la 

beef broth 

sterile tube 

not 

lb 

milk 

plugged 

heated 

2a 

beef broth 

unsterilized 

not plugged 

2b 

milk 

tubes 

not heated 

3a 

beef broth 

unsterilized 

heat to 100° C. 

3b 

milk 

tubes, plugged 

for ten minutes 

4a 

beef broth 

unsterilized 

heated to 120° C. 

4b 

milk 

tubes, plugged 

for 30 minutes 

5a 

beef broth 

unsterilized 

heat to 120° C. 

5b 

milk 

tubes, plugged 

and inoculate 


2. Treat tubes 3a and 3b in a cup of boiling water 10 minutes over the 
Bunsen flame. 

3. Prepare a labeled piece of paper giving the experiment and desk number. 
Fasten this paper around the tubes 4a and 4b, and 5a and 5b with a rubber 
band. Place them in the autoclav to be heated for 30 minutes at 116°-120°C. 



FIG ll-CORRECT METHOD OF HOLDING TUBES WHEN TRANSFERRING CULTURES 
Note how the plugs are held between the fingers of the right hand. The plugs ..re never laid down- 
The tubes are held as nearly horizontal as possible. 

































17 

4. When cool, tubes 5a and 5b should be inoculated with a loopful of soil 
or tap water. (See Fig. 11.) 

5. Examine the tubes at each opportunity for two weeks. Tabulate the 
data as shown in Table I on the regular note paper. 

6. At the end of the observation period stain some of the milk and broth 
from tubes 4a and 4b, and 5a and 5b. Use carbol fuchsin, one minute for the 
broth, and methylene blue three minutes for the milk. 


TABLE I.—Sterilization of Milk and Broth 


Tube 

No. 

Treatment 

of 

beef broth 

Appearance 
after 
two days 

Appearance 
after 
one week 

Appearance 

after 

two weeks 

la 

2a 





3a 

4a 





5a 





Tube 

No. 

Treatment 

of 

milk 

Appearance 
after 
two days 

Appearance 
after 
one week 

Appearance 

after 

two weeks 

lb 

2b 





3b 

4b 





5b 






7. Describe efficient methods for the sterilization of milk and broth. 

(a) What was the macroscopic evidence that any milk or broth 
tubes were not sterile? Microscopic? 

(b) What does “sterile” mean? Which tubes were sterile? 

(c) Why is an autoclav temperature of 116° C more efficient than 
hot air oven temperature of 150° C? 

(d) Give a satisfactory method of sterilizing water, milk, Petri 
dishes, bandages, a room. 






















18 


Exercise 8. The Distribution of Bacteria 
MATERIALS: 

A case of sterile pipettes 
One sterile, empty test tube 
Five sterile Petri dishes 

Five tubes of sterile, meat extract-peptone agar 
Soil suspension 

1. Label the five dishes near the edge of the covers. Be very careful not 
to handle the dishes any more than is necessary and remove the covers only 
when necessary. 

2. Mark the labels as follows: 

(а) “Air” 

(б) “Finger tips” 

(c) “Sterile” 

(d) “Soil suspension, onb drop” 

(e) “Tap water, one cubic centimeter” 

3. With a sterile pipette place a drop of the soil suspension in dish (d). 

4. Collect some tap water in a sterile test tube. 

5. With another pipette place one cc. of tap water in dish (e). 

6. Melt five tubes of agar (bacterial food in a jelly) in the metal cup by 
boiling them in water. When the agar is melted, cool to 45°C. by mixing cold 
water carefully with the hot water in the cup. Great care must be exercised 
not to cool the water below 45°C. At this temperature bacteria can be mixed 
with culture media without injuring them. 



FIG. 12—THE CORRECT METHOD OF POURING LIQUEFIED CULTURE MEDIA INTO 

PETRI DISHES 

After wiping the water from the outside of a tube of agar, remove its plug, 
flame the mouth of the tube in the gas flame and pour the agar into dish (a), 
(See Fig. 12). Tip the dish carefully from side to side to spread the agar 
over the entire bottom of the dish. Place the dish on a level part of the 
desk until the agar becomes solidified. In the same way pour agar into the 
other dishes. 

7. Expose dish (a) with the cover off to the air of the laboratory for 
twenty or thirty minutes. Leave the cover right side up by the side of the dish 
during the exposure. 

8. Carefully touch the finger tips and nails of one hand to the surface of 
the agar in dish (b). Keep dish (c) closed. 

9. When the agar is solid and dish (a) is exposed, invert the plates in the 
28°C incubator for one week. 

10. Rinse out pipettes used and leave on the desk to be sterilized. Wash 
tubes that contained agar, and invert in the desk. 






19 


11. Examine the plate cultures. The differently colored and shaped spots 
are colonies of bacteria. Where one colony is separate from the others, uni- 
from in shape, color, and appearance, it is probably a colony or group of 
organisms that has grown from a single cell. 

Select three colonies with the instructor’s approval and mark them 1, 2, 3 
respectively with a pen or with a wax pencil. 

12. Examine them with the 16 m.m. objective. Place the Petri dish, in¬ 
verted, cover and all, on the stage of the microscope. Draw and describe 
them according to the outline in Appendix A. 

(a) What is the difference in appearance of the agar in the dishes, 

(1) as compared to the time when poured into the dishes; 

(2) as compared to each other after one week? 

( b ) Plow may the appearance of the dish labeled “sterile” be 
explained? 

(c) What is the advantage of plate cultures? 

( d ) Who first introduced the plate culture method? 

( e) Why do colonies not form in liquid media? 

(0 Is it always true that each colony forms from one cell? Give 
reasons for your answer. 


20 


Exercise 9 Isolation of Pure Cultures of Bacteria 
MATERIALS: 

3 tubes of plain agar for slopes 
3 tubes of plain broth 

1. Pure cultures are to be made from the colonies described in Exercise 8. 
A pure culture of a bacterium, yeast or mold is a culture which contains only 
one kind of a bacterium, yeast or mold. It is assumed that each of the three 
colonies selected have originated from a single organism. 

2. Mark three tubes of agar, 1, 2, 3 respectively. If the agar is not sloped 
in the tubes, melt and slope it as shown by the instructor. 



FIG. 13—“FISHING” OFF COLONIES FROM A PETRI DISH CULTURE 


3. When the agar has become firm, inoculate tube No. 1 from colony No. 
1 (Exercise 8) as follows: Heat the striaght wire needle in the flame to red¬ 
ness to sterilize it. When cool, touch the tip of the needle to the colony 




























21 


(Figs. 13 and 14) and spread the adherent organisms on the agar in the tube 
by drawing the needle once lightly over the surface of the agar from the 
bottom to the top. Be careful that the needle does not touch any other 
colony during the operation. Inoculate the other two tubes of agar from the 
respective colonies. 

4. Inoculate the broth tubes from the same colonies, rinsing the organisms 
from the needle in the broth. 

5. Leave the cultures in the incubator for 48 hours or in the locker for four 
or five days. 

6. Examine the cultures. Look for a streak along the needle track on the 
agar slopes, and for turbidity, sediment, or a surface film in the broth cultures. 
Draw and describe the cultures according to the outline for broth and agar 
streak cultures in Appendix A. 

7. Examine the organisms in each broth culture by means of the hanging 
drop and determine if they are motile. Record the morphology of the or¬ 
ganisms in each culture. 

8. (a) What are some other kinds of agar tube cultures besides slant 

cultures? 

( b ) What are the advantages of solid media for tube cultures as com¬ 
pared to liquid media. 


















Exercise 10. The Microscopic Examination of Bacteria 
in Pure Culture 

MATERIALS: 

3 plain agar slants 

Pure cultures made in Exercise 9 

9 plain slides 

1. Carefully label three plain agar slants and make new transfers of the pure 
cultures isolated in Exercise 8. Place them in the incubator for 48 hours. 
Save them for use in Ex. 11. 

2. Carefully clean 8 slides. Arrange three of them in a row and label each 
of the three the same as one of the pure cultures that have been isolated. 
Place a small drop of water, about 1 /8 inch across, in the center of each slide. 
Remove some of the growth from the agar culture with the sterile wire needle 
and mix it with the water on each slide, spreading the mixture as thin as 
possible. (See Fig. 8.) Permit each to air dry. Cause the bacteria to adhere 
to the slides by passing them three times through the top of the gas flame 
(fixing). (See Fig. 9.) Lay the slide upon the wire cover of the sink for stain¬ 
ing. (See Fig. 10.) Repeat with each of the other cultures. 

3. Stain one slide from each culture with methylene blue for 5 minutes. 
Stain another slide from each culture with carbol fuchsin for one minute. 
Stain the remaining slide from each culture by means of the Gram method as 
follows: 

(a) Stain with ammonium oxalate crystal violet for 2 or 3 minutes. 

( b ) Pour off the stain. 

(c) Do not wash. 

( d ) Treat with Gram’s iodin (Lugol’s) solution for one minute. 
Pour off and treat again for one minute. 

(e) Pour off iodine. Wash in water quickly. Blot off excess 
water quickly but do not permit the film to air dry. 

(0 Decolorize in equal parts of acetone and 95 per cent ethyl 

alcohol, until the stain no longer washes from the slide. 

(g) Dry between blotters. 

(/?) Counter stain one minute with aqueous one percent safranin. 

(i) Wash and dry between blotters or air dry. 

This method of staining is an aid in the classification of bacteria. Some 
bacteria do not lose the purple color imparted to them by the crystal violet 
when treated by this method; others do lose the color. The former are termed 
Gram-positive, the latter Gram-negative organisms. 

4. Examine the slides. Draw and describe one slide from each culture. 

(a) What is the form of the organisms from each culture? (Fig. 5.) 

(b) Are any of them yeasts? 

(c) Do all of the organisms stain by the Gram method? 

(</) Are there spores in any of the cultures? 

(e) What stain shows each organism best? 

(f) Under the microscope, how can bacteria be distinguished from 
yeast? 

(g) In a Petri dish culture there is a mixture of bacteria, yeast, 
and mold colonies. What should be done and what would be 
found in distinguishing them? 






























23 


Exercise 11. Enzyme Production by Microorganisms 

MATERIALS: 

Garden soil suspension 
5 tubes of plain agar 
5 tubes of starch agar 
5 tubes of sterile milk 
4 tubes of sterile gelatin 
Pure cultures of bacteria 
Sterile pipettes 
10 sterile Petri dishes 

A. Carbohydrate Hydrolyzing Enzymes icarbohydrases) 

Amylase. 

1. Melt the starch agar and cool to 45° C. 

2. Label five sterile Petri dishes as follows: 

(a) Starch agar, soil suspension. 

(b) Starch agar, pure culture 1. 

(r) Starch agar, pure culture 2. 

( d) Starch agar, pure culture 3. 

(e) Starch agar, B. graveolens culture. 

3. By means of a sterile pipette place a drop of soil suspension in Petri 
dish (a). 

4. Immediately pour starch agar into all of the Petri dishes being care¬ 
ful to observe the precautions indicated in Exercise 8. 

5. When the agar is solid inoculate the four remaining dishes with the 
organisms indicated. Use the straight needle and make the inocula¬ 
tion at one spot in the center of the dish. 

6. Incubate the cultures inverted at room temperature for 48 hours. 

B. Protein Hydrolyzing Enzymes (protelnases) . 

Caseinase 

1. Label five Petri dishes as follows: 

(a) Milk agar, soil suspension. 

( b ) Milk agar, pure culture 1. 

(c) Milk agar, pure culture 2. 

( d) Milk agar, pure culture 3. 

(e) Milk agar, B. graveolens culture. 

2. With a sterile pipette, place one cc. of sterile milk in each of the 
Petri dishes. 

3. By means of a sterile pipette place a drop of soil suspension in Petri 
dish (a). 

4. Melt five tubes of plain agar and cool to 45° C. 

5. Immediately pour agar into all of the Petri dishes being careful to 
mix agar and milk until the whole has a uniform white appearance. 

6. When the agar is solid, inoculate the agar at one point in the center 
of each dish. Use the straight transfer needle, and make inoculations 
as designated in paragraph A5. 

7. Incubate the cultures inverted at room temperature for 48 hours. 


24 


Gelatinase 

1. Prepare stab cultures in gelatin using the pure cultures 1, 2, and 3 from 
Exercise 10 and the culture of B. graveolens. 

2. Be sure the gelatin is solid. If it has the slightest tendency to become 
soft, the tubes should be placed in a glass of ice-water until solid. 

3. Make the stab culture in the gelatin by thrusting the inoculated straight 
wire into the center of the solid medium, nearly to the bottom of the test tube. 
Be sure the wire is perfectly straight. 

4. Incubate the cultures in the 20° C. incubator to prevent melting of the 
gelatin. Examine after one week. 

Observation of Cultures 

1. Flood the starch agar plates evenly with Lugol’s solution and drain dry. 

2. Examine both the starch agar and milk agar plates for halos around the 
colonies. Notice the clear, yellow or wine red color of the halos in starch agar. 

3. Examine, draw and describe the gelatin stab cultures. See Appendix A, 
Part B, pages 77 to 80. 

(a) What causes the clear halos around the colonies? 

( b) How do you account for the enzyme action so far from the 
colonies in some instances? 

(c) What is the value of these enzymes to the bacteria? 

( d ) How are these bacterial enzymes of value to man? 

(e) Were any molds observed to produce enzymes? 

(0 What are the common names applied to the enzyme changing 
starch to maltose? 

( g ) What kind of chemical change is produced by the enzymes 
studied in the exercise? 

( h ) Name three other types of changes caused by enzymes? 

( i ) State your conception of an enzyme? 

O') Why is agar used for streak cultures more than gelatin? 

( k) Why is gelatin preferred to agar for stab cultures? 




25 


Exercise 12. The Fermentation of Carbohydrates by Bacteria 
MATERIALS: 

2 fermentation tubes of dextrose broth containing brom cresol 
purple 

2 Durham tubes of lactose broth containing brom cresol purple 
1 culture each of Streptococus lactis and Aerobacter aerogenes 

1. Inoculate one Durham tube of lactose and a fermentation tube of 
dextrose broth with each of the organisms furnished. 

2. Place the cultures in the incubator and examine them in 48 hours. 

3. Observe the reaction of the broth in the tubes. Note whether gas has 
been formed in both lactose and dextrose broth. If gas has been formed in the 
fermentation tubes, measure it and determine its composition as shown by 
the instructor. 

4. Compare the color of the indicator in the Durham tubes with a set of 
standard solutions containing brom cresol purple. Record the change in reac¬ 
tion as shown on the pH scale of hydrogen ion concentrations. (See Fig. 15.) 

(а) What is the range in pH of brom cresol purple? 

(б) What was the change in the reaction of the sugar broths 
where the bacteria grew? (See the diagram Fig. 15). 

(c) What acids do bacteria most commonly form from dextrose 
and lactose? 

( d ) What was the gas formed in the fermentation tube? 

(e) What gaseous substances are usually formed in the decom¬ 
position of carbohydrates? 

( f ) What are the steps in the conversion of an insoluble carbo¬ 
hydrate like starch into acid by bacteria? 

(g) Why is the bacterial decomposition of carbohydrates of more 
industrial importance that that caused by molds? 

(/?) What is the advantage to bacteria of being able to ferment 
carbohydrates? 

(0 How does the action of yeast on sugars differ from the action 
of bacteria? 



KC1 


GASTRIC JUICE 
ASPERGILLUS 
LIMIT 

YEAST LIMIT 
WINES 


SILAGE 


ESCHERICHIA 
COLI LIMIT 


MILK 

NEUTRALITY 

BLOOD 


BORAX 


*NH 4 0H 


FIG. 15—D IA G R A M M A T I C 
REPRESENTATION OF THE 
pH SCALE OF TRUE ACIDITY 


The pH values, given as the log of 
-, are shown on the numbered 

horizontal lines. The acid end is at 
the top, the alkaline at the bottom. 
The diagonal line shows the pro¬ 
portion of H ions to OH ion 3 at each 

TT 

pH. Thus neutrality whe:e 577 -= 1 

H.O 


is at pH 7.0. The true acidity given 
in pH values for some familiar sub¬ 
stances is indicated The working 
range of brom-cresol purple is shown 
by the curved line. 


ALKALINE 






























26 


Exercise 13. The Production of Pigment by Bacteria 
MATERIALS: 

6 plain agar slants 
6 glucose agar slants 
3 plain gelatin for stab cultures 
3 glucose gelatin for stab cultures 

One culture each of Ps. prodigiosus, Ps. flurorescens and Aero- 
bacter aerogenes. 

1. Prepare one plain gelatin and one glucose gelatin stab culture from each 
of the cultures as follows: Make sure the transfer needle is straight. Sterilize 
the needle, cool, and touch the tip to the bacterial growth. Carefully make a 
stab with the inoculated needle in the center of the gelatin. The stab should 
be made parallel to the sides of the tube, and within a few millimeters of the 
bottom. 

2. Place the three cultures, labeled and held together by means of a rubber 
band, in ice-water until they can be placed in the 20°C incubator. 

3. Prepare two plain and two glucose agar slant cultures of each of the 
organisms. 

4. Place one plain agar slant and one glucose agar slant of each culture at 
room temperature. Incubate the other plain and glucose agar slants at 37°C. 

5. Examine the cultures every 48 hours for one week. 

6. At the end of a week record, in table form, the absence of pigment as — 
and different degrees of pigment as +, + -f, or + + +. Brightness of pigment 
should be considered and not the amount of growth. 

7. Place a few cc. of each of distilled water, chloroform, and carbon 
bisulphide in small test tubes. Add a large loopfull of the brightest pigmented 
culture to each. Observe any solubility in the solution. 

8. (a) Where in the cultures is the pigment seen most conspicuously? 

( b ) How may this be explained? 

(c) What composition of the medium is the most favorable for 
pigment production? 

(d) What temperature is most favorable for pigment production? 

(e) What does this suggest about the habitats of the organisms? 

(0 What is the relation of oxygen to pigment production? 

(g) What cultures showed this? 

{h) What pigments are water soluble? 

(i) What are pigment-producing organisms called? 

(J) What is the practical agricultural significance of pigment- 
producing organisms? 



27 


Exercise 14. The Influence of the Composition of the Sub¬ 
stance on the Types of Fermentation that will Occur in It. 

MATERIALS: 

4 100 cc. portions of sterile broth 
8 sterile test tubes 
1 300 cc. flask of sterile sand 
4 sterile Petri dishes 
N/10 sulphuric acid 
Sterile glucose solution 

Pure cultures of Psuedomonas fluoresce ns, Saccharomyces 
cerevisiae and Aspergillus species. 

1. Treat four 100 cc. portions of sterile broth, pH 7.0 as follows: 

No. 1. Leave as it is. 

No. 2. Add 5 cc. of 20 per cent glucose solution. 

No. 3. Add 1 cc of N/10 sulphuric acid, giving a reaction of ap¬ 
proximately pH 4.5. 

No. 4. Add glucose and acid. 

2. Mix well with gentle shaking; and carefully pour two sterile test tubes, 
one-third full from each flask. Be sure the tubes are labeled properly. 

3. Pour each of four sterile Petri dishes one-third full of sterile sand. 

4. Carefully label the dishes and moisten the sterile sand to saturation 
with one of the broths prepared above. 

5. Inoculate the tubes and dishes as follows: 


Broth No. 

Tul 

bes 

Dishes 

1 

1 

2 

Mold 

Bacteria 

Yeast 

2 

Bacteria 

Yeast 

Mold 

3 

Bacteria 

Yeast 

Mold 

4 

Bacteria 

Yeast 

Mold 


Use a culture of Psuedomonas fluorescens for the bacteria, Saccharomyces 
cerevisiae for the yeast and Aspergillus for the mold. 

6. Incubate the cultures in the desk for two weeks. Examine them at 
the end of the first and second weeks for signs of growth. Construct a table 
showing the type of fermentation that has taken place in each case. Record 
the presence of gas, odor and the reaction. 

7. (a) What do the results of this exercise indicate in regard to the con¬ 

ditions favoring and opposing the growth of bacteria, yeasts, 
and molds? 

(b) Construct a diagram of the pH range of hydrogen ion concentra¬ 
tion showing the optima for bacteria, yeasts, and molds? 
(See Fig. 15). 













28 


(c) The composition of milk, broth, grape-juice, and lemon juice is as 
follows: 


MILK 


Water 


S7.10 per cent 

Casein and Albumen 


3.40 “ 

• • 

Fat 


3.90 “ 

“ 

Milk Sugar (lactose) 


4.75 “ 

“ 

Ash constituents 


0.75 “ 

•• 

Reaction pH 

MEAT BROTH 

6.S 


Water 


98.70 “ 

“ 

Peptone 


1.00 “ 

«• 

Meat Extract 


0.30 “ 

“ 

Reaction pH 

GRAPE JUICE. 

7.h 


Water 


82.90 “ 

“ 

Sugar (dextrose) 


15.00 “ 

“ 

Tartaric Acid 


1.00 “ 


Soluble Nitrogen 


0.40 “ 

“ 

Ash 


0.70 “ 

•• 

Reaction pH 

LEMON JUICE 

4.50 


Water 


94.30 “ 

“ 

Sugar 


0 20 “ 

“ 

Citric Acid 


500 4 

“ 

Ash 


0.50 “ 

“ 

Reaction pH 


2.20 



What determines the changes and the kinds of organisms causing the 
changes in the above materials? 

( d ) Why does milk undergo an acid fermentation? 

( e ) What is the source of the acid in the lemon and grape juice? 
(0 What kind of change took place in the broth? Why? 

(g) What characteristics of natural organic substances determine 
the possiblity of their decomposition by microorganisms? 
(/?) What relation do these facts bear to agriculture? 



% 


PART III 


SOIL BACTERIOLOGY 

Exercise 15. The Number and Kinds of Bacteria in 
Different Soils 

MATERIALS: 

9 tubes sodium caseinate agar 
9 sterile Petri dishes 
Case sterile pipettes 
3 9 cc. sterile water blanks 
1 99 cc. sterile water blank 

Suspension of soils, 1 gram of soil in 1000 cc. of sterile water 

There are enormous numbers of bacteria in most fertile soils, and on this 
account a very small amount of the soil must be used in determining the num¬ 
ber of bacteria. Results of determinations are always given per gram of soil, 
usually calculated on a “dry” basis. In all of the following work two repre¬ 
sentative soils will be used. One sandy loam, “soil A,” and one black garden 
soil, “soil B,” Prepare cultures from one soil. 

1 . Observe that there are 1 to 1000 dilutions of the two soils on the desk. 

2 . Prepare 4 quantitative dilutions of one soil and make duplicate plate 
cultures with 1-1000, 1-10,000, 1-100,000, and 1-1,000,000 gram of soil. 
Prepare a “blank” without the soil. Label dishes with the name of the soil 
and the dilution used. 



FIG. 16—MANNER OF MAKING THE SOIL DILUTIONS 

3. Proceed by arranging the water blanks and Petri dishes as shown in Fig. 
16. Make all transfers with sterile 1 cc. pipettes as indicated by the continu¬ 
ous arrows, and transfer 1 cc. each time. All steps marked “A” are to be 
carried out with the first pipette and in the order given; steps marked “B” 
with the second pipette, etc. 

4. Shake the 1 to 1,000 gram dilution well with a rotary motion, at least 
fifteen times. While the liquid is still in motion, transfer 1 cc. to the necessary 























30 


water blank or Petri dish. Be sure each dish receives the dilution for which it 
is labeled. Prepare the blank as shown. 

5. Melt 9 tubes of plain agar and cool them to 45° C. 

6 . Pour the plates, being very careful to get the dilution water well mixed 
with the agar. Place the poured plates on a level surface while the agar 
hardens. Rinse out the tubes with hot water while; the agar remaining in 
them is still soft. 

7. When the agar is solid, incubate the dishes at room temperature. Ex¬ 
amine the plates in 4 to 6 days. 

8 . Assuming that each colony has grown from a single organism, count the 
colonies on the best plates and estimate the number of organisms per gram of 
soil. 

Plates containing between 70 and 200 colonies give the most accurate 
results and incidentally are the most easily counted. 

9. Record the results on the regular note paper in tabular form like the 
following: 


TABLE II.—Bacterial Content of Soil 


Dilution 

Number of 
Colonies 
on Plate 

Number o^ 
Bacteria 
Per Gram 
Average 
of two 
plates 

Apparent 
Number of 
Kinds from 
Gross 
Appear¬ 
ance 

Number of 
Molds 

1/1000 g. 






1 /10,000 g. 






1 /100,000 g. 






1 /1,000,000 g. 






Blank 






(a) From the results, can you say that there are many bacteria in 
average soils? 

( b ) Are there many kinds present? Did all of the organisms 
present develop? Why? Give two good reasons for the an¬ 
swer. 






























31 


(c) Were there any organisms developing in the dish marked 
“blank”? What does this show? 

(d) How do the number and kinds of organisms in the soil ex¬ 
amined compare with the results of the examination of the 
other soil? 

















































* 

. • ^ 




. • 


' 


f 

' 





g 



























































32 


Exercise 16. Ammonification by Soil Bacteria 
MATERIALS: 

3 tubes of beef broth 
3 tubes of casein solution 
3 tubes of urea solution 
Soil samples 

The protein of plant and animal tissues and urea (CO(NH 2 ) 2 ), in which 
compound a great deal of the nitrogen in urine is found, readily undergo 
ammonification. Since carbon dioxide (C0 2 ) is practically always present in 
decomposing materials, the ammonia formed unites with it to form ammonium 
carbonate (NH 4 ) 2 G0 3 ). 

1. Label each of the 3 tubes of beef broth, casein and urea medium with the 
name of medium, and the soil with which it is to be inoculated. 

2 . Inoculate one of each three tubes with a small quantity of one of the 
soils used in exercise 15, by moistening the wire loop with tap water and 
thrusting it into the soil. The amount of soil that will adhere to the moist loop 
is sufficient. Another tube of each three is to be inoculated with the other soil. 
The remaining tube is left without inoculation and is to serve as a control. 

3. After 48 hours incubation at room temperature, test the inoculated tubes 
for ammonia by placing a drop of Nessler solution in a depression on a spot 
plate and adding to it one loopful of the culture. The presence of ammonia 
is indicated by the development of a color, a faint yellow with small amounts 
of ammonia, a deep yellow or brown with larger quantities and a brownish 
precipitate with still larger quantities. 

4. After incubation for one week test both the inoculated and control tubes 
for ammonia. 

5. Record the results of both tests on the regular note paper as in Table III. 
Rank the solutions according to the amount of ammonia present, the solution 
containing the smallest amount being ranked 1. 


TABLE III.—Formation of Ammonia by Soil Bacteria 



Rank afte 

r 48 hours 

Rank aft< 

?r 1 week 

Control 

soil A 

soil B 

soil A 

soil B 

Beef Broth 






0 

Urea 






Casein 







6 . In recording the conclusions, answer the following questions: 

(a) From the above data, what is to be concluded as to the cause 
of ammonia formation in soils? Why? 

( b ) What was in the casein solution and beef broth that yielded 
ammonia upon decomposition? 

(c) Give the most common sources of such substances in farm 
practice. 















33 

(</) Do the ammonifying organisms render nitrogen available to 
plants as food? Explain. 

(e) From which one of the substances would nitrogen be made 
most quickly available to the green plant? Why? 

(0 In which of the tubes is ammonia made evident by odor? 


/ 










. : ■. 











-. 



I 

. •. . * i 




! 


. 


' 






• • . - 









































/ • 


* , • 






-* - 

. 

%■ 




























Exercise 17. Nitrification 


MATERIALS: 

2 100 cc. Erlenmeyer flasks 

4 one-fourth pint milk bottles 

Ammonia and nitrite media 

25 cc. graduate 

Soil samples 

Milk bottle caps 

Cotton 

Nitrification includes the oxidization of ammonia (NH„) to nitrites (N o 0 3 ) 
and their subsequent oxidization to nitrates (N 3 0 5 ). The organisms which 
carry out the oxidization of ammonia to nitrites and nitrites to nitrates are 
present in most cultivated soils. Their activity in a given soil may vary with 
the cultivation and cropping of the soil. 

The solutions used for the cultivation of these organisms contain no organic 
matter—only in organic salts. 

A. Nitrite Formation 

In the culture medium for the nitrite-forming organisms, the source of 
nitrogen is ammonium sulphate (NH 4 ) 2 S0 4 and in the beginning thesolution 


contains no nitrites or nitrates. 

Ammonium sulphate (NH 4 ) 2 S0 4 )_ 2.00 grams 

Di-basic potassium phosphate (K,HP0 4 ) __ 1.00 “ 

Magnesium sulphate (MgS0 4 )_ 0.50 “ 

Ferrous sulphate (FeS0 4 )_ trace 

Sodium chloride (NaCl)_ 0.40 “ 

Water_1000.00 “ 

Magnesium carbonate (MgC0 3 )_ excess 

1. See that 3 milk bottles are clean 


2. The culture solution is on the desk in the white enamel graduate and is 
labeled “ammonia medium.” 

Stir thoroughly until the undissolved substances are in uniform suspension. 
Transfer by means of the 25 cc. graduated cylinder enough (about lOcc.) of the 
culture solution to each milk bottle and to the flask to form a layer of about 
one-fourth inch deep. 

3. Inoculate one bottle with 2 or 3 measurefuls of soil A and another 
with 2 or 3 measurefuls of soil B. Inoculate the solution in the flask with 
both soils. Label the flask in pencil on the etched portion. Be sure the label 
states the desk number as well as the notations about the experiment. 

4. Leave the flask properly plugged and labeled on the desk to be sterilized. 

5. Test each culture and the control once a week for: 

(a) Ammonia 

( b ) Nitrites 

When any culture no longer gives a positive test for nitrites, test it for 
nitrates. 

B. Nitrate Formation 

In the culture medium for the nitrate-forming organisms, the source of 
nitrogen is nitrite and the solution contains no nitrates at the beginning. 








t 




35 


Sodium nitrite (NaN0 8 ). _______1.00 gram 

Di-basic potassium phosphate (K 2 HP0 4 )_ __1.00gram 

Magnesium sulphate (MgSOj __0.30 gram 

Sodium carbonate (anhydrous) (Na 2 C0 3 )_1.00 gram 

Sodium chloride (NaCl). . ______0.50 gram 

Ferrous sulphate (FeS0 4 )_ trace 

Water-„. 1000 


1. Place in each of two clean bottles and a flask enough of the solution 
marked nitrite medium to form a layer one-fourth inch deep. Inoculate the 
bottles and flask as described in paragraph 3 above. 

2. Incubate all the cultures in the desk. 

3. Test the cultures once each week for: 

(a) Nitrites. 

(b) When Trommsdorf’s solution no longer gives a test for 
nitrites, test for nitrates. 


Note—To test for ammonia. —Place in a depression on a spot plate one 
drop of Nessler’s solution. Touch with a sterile glass rod carrying 
a drop of the solution to be tested. Do not stir. A test gives a 
brown color. 

To test for nitrites. —Place in a depression on a spot plate, 
Trommsdorf’s solution one or two drops and 1:3 sulphuric acid 
two or three drops. Mix. Touch with a drop of the solution to 
be tested. Do not mix. 

A test gives a deep blue color. 


4. 


To test for nitrates. —Place in a depression on a spot plate one 
drop of a solution of diphenylamine in concentrated sulphuric 
acid. Touch with a drop of the solution to be tested. 

A test gives a very dark blue color. Nitrites also give a blue 
color with diphenylamine, therefore before one can be certain 
nitrates are present, the solution must be tested for nitrites by 
the Trommsdorf reagent and the absence of nitrites shown. 

(a) What do you conclude regarding the nitrifying power of the 
two soils studied? 

( b ) What agricultural practice tends to promote the activity of 
these organisms in the soil? Why? 

(c) What is the purpose of the magnesium carbonate in the 
solution containing ammonium sulphate? 

(d) What use does the nitrite-forming organism make of the 
ammonium sulphate? 

(e) What use does the nitrate-forming organisms make of the 
sodium carbonate? 

(0 Why is plenty of air necessary for the growth of these 
organisms? 

(g) Who first isolated pure cultures of these organisms? 

(. h ) What are the names of the two species of organisms concerned 
in these processes? 

(j) Why is it not necessary to add magnesium carbonate to the 
solution containing sodium nitrite? 

















36 


TABLE IV.—Nitrification by Soil Bacteria 


Tim 

in 

Weeks 

Ammonia Medium 

Nitrite Medium 

Soil A 

Soil B 

Control 

Soil A 

Soil B 

Control 

NHs 

NzOs 

N2O5 

NHs 

N2O3 

N2O5 

NHs 

N2O3 

N2O5 

N2O3 

N2O5 

n 2 o 3 

N2O5 

N2O3 

N2O5 

1 







* 









2 
















3 












1 




4 
















5 
















6 












1 





(J) How do these organims differ from those already studied, viz: 
those that grew on the soil, dust and milk plates, in regard 
to the nature of their food supply? 

(k) In what part of a farm sewage disposal system consisting of a 
septic tank and drains, do these organisms function? 

(/) In large municipal sewage disposal plants, how are these or¬ 
ganisms helped in their work by special mechanical devices? 










































37 


Exercise 18. Denitrification 

MATERIALS: 

5 tubes of Giltay’s solution 
1 fermentation tube of nitrate broth 
Soil samples 
Horse manure 
A culture Ps. pyocyaneus 

If there is an accumulation of nitrates in the soil and anaerobic conditions 
are established by the soil becoming saturated with water, in the presence of 
large amounts of organic matter loss of nitrogen may result. This loss of 
nitrogen is due to the action of anaerobic organisms which reduce nitrates. 

The nitrates may be reduced to nitrites only, or completely to free gaseous 
nitrogen. In the latter case there is an actual loss of soil fertility. 

1. Inoculate 5 tubes of Giltay’s solution as follows: 

1 tube with soil A 
1 tube with soil B 
1 tube with horse manure 

1 tube with a pure culture of Pseudomonas pyocyaneus 
1 tube leave sterile and uninoculated and label “sterile control.” 


giltay’s solution 

Water__1000.00 grams 

Potassium nitrate (KNO a )_ 2.00 “ 

Asparagin (C 4 H 8 N 8 0 3 )_ 1.00 “ 

Magnesium sulphate (MgS0 4 )_ 2.00 “ 

Calcium citrate (Ca 3 (C 6 H 5 0 7 ) 2 -f4H 8 0)_ 8.00 

Monobasic potassium phosphate (KH 2 P0 4 ) 2.00 

Calcium chloride (CaCl 8 )_ 0.20 

Iron chloride (FeCl a )_ 0.20 

2. Inoculate the fermentation tube of nitrate broth with horse manure. 
Use about one gram. 


3. Place all the cultures in the incubator for two days or one week. 

4. Test the test tube cultures first for nitrites, and if nitrites are absent, 
then test them for nitrates. 

5. Test the fermentation tube culture in the same way and if gas is present 
determine its composition as was done in Exercise 12. 

6. Record the results on the regular note paper as shown in table V. 

(a) What changes have taken place in the fermentation tube 
culture? 

( b) What has happened? 

(c) How are the changes to be explained? 

(d) What changes do the tests indicate? 

(e) Under what kind of conditions did the changes come about? 

(f) In Giltay’s solution, what substances are food for energy and 
what food for growth? 

(g) In the main, what are the changes produced in Giltay’s 
solution by the bacteria? 

( h) Where do the organisms get their oxygen? 






























♦ 








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^ I 




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* * 












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l r ‘ 1 












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38 


TABLE V.—Denitrification by Soil and Manure Bacteria 


Source of 
bacteria 

Medium 

Gas 

Formation 

Nitrites 

Nitrates 

Soil A 





Soil B 





Horse 

manure 





Denitrifying 

organism 





Sterile 

control 






(i) How do conditions for denitrification come about in agricul¬ 
tural practice? 


















39 


Exercise 19. Free Nitrogen Fixation 
Noil-symbiotic Type 

MATERIALS: 

2 one-fourth pint milk bottles and caps 
25 cc. graduate 
Nitrogen-free liquid medium 
Soil samples 

Pure cultue of Azotobacter 

Many soils upon which legumes are not growing, and to which nitrogen is 
not added in the form of chemical compounds or fertilizers of any kind, still 
have the power of materially increasing their nitrogen content. This increase 
is due to the presence in the soil of species of free nitrogen-fixing bacteria. 
These organisms are capable of maintaining an existence and of fixing nitro¬ 
gen independent of higher plants. 

The organisms that have the power to take the free nitrogen of the air and 
combine it in their cells can grow in a nitrogen-free culture solution. This 
culture medium contains only inorganic salts in solution and some simple 
carbohydrate or similar carbon compound which contains no nitrogen, e. g., 
the sugars, some of the alcohols, as mannite, etc. Such a solution is prepared 


as follows: 

Mannit (C 6 H 8 (OH c )_ 15.00 grams 

Magnesium sulphate (MgS0 4 )_ 0.20 “ 

Sodium chloride (NaCl)_ 0.20 “ 

Calcium sulphate (CaS0 4 )_ 0.10 “ 

Calcium carbonate (CaC0 3 )_ 5.00 “ 

Dibasic potassium phosphate (K 2 HP0 4 )_ 0.20 “ 

Water_'_1000.00 “ 


1. The above culture medium is on the desk. With the graduate place 
25 cc. of it in each of the two milk bottles. Be sure to stir the medium well 
before measuring any of it, as the insoluble substances settle to the bottom. 

2. Label the bottles. Inoculate one with two or 3 measures of soil A and 
the other with the same amount of soil B. Cap the bottles. 

3. Place the bottles in the incubator at 25° C. where they will not be dis¬ 
turbed. Watch for the development of a white, gelatinous film which is very 
conspicuous and soon turns brown or black. 

4. Make a microscopic preparation from the film on the surface of each 
solution, according to the following method: Place a drop of strong iodine 
solution upon a clean slide. Place some of the organisms from the film in the 
iodine solution. Carefully cover with a cover glass. The organisms will float 
in the solution under the cover glass. 

5. Examine the preparation with the 4 m. m. objective. The large oval or 
coccoid bodies are some species of the free nitrogen-fixing organism, Azotobac¬ 
ter. Long, rather oval or mace-shaped organisms, which have a blue appear¬ 
ance, are sometimes seen; they are the anaerobic species of free nitrogen-fix¬ 
ing organisms, Clostridium butyricum. 

6. Examine the agar slope culture of Azotobacter furnished. Draw and 
describe it according to the outline for study of species. See Appendix A. 















40 


7. Make one microscopic preparation from the agar and stain the organ- 
sims with methylene blue. Draw and describe the organisms as seen with the 
oil-immersion objective. 

8. The relative number of these organisms is soils A and B can be judged by 
the rate and extent of the development of the brown film. 

(a) What is the shape of the Azotobacter^. 

(b) Is the organism motile? 

(c) How does it compare in size with other bacteria? 

( d ) What organisms have you studied of which it reminds you? 

(e) Is the organism aerobic or anaerobic? 

(0 What is the value of Azotobacter to the soil? 

(g) Where did the nitrogen come from that is in the film on the 
liquid in the bottles? 

(h ) What use does Azotobacter make of the mannite? 

(0 What purpose is served by the calcium carbonate? 




41 


Exercise 20. Effect of Variation in Food on the Growth 
of Azotobacter 

MATERIALS: 

2 tubes of nitrogen-free agar 
2 tubes of same agar without sucrose 

2 tubes of the same agar with lactose in place of sucrose 
2 tubes of the same agar without phosphate 
1 9 c. c. sterile water blank 
Pure culture of Azotobacter 

1. Prepare a heavy suspension of organisms from the pure culture by trans¬ 
ferring them to the sterile water. Shake well until they are evenly distributed. 

2. After carefully labeling the agar slopes, inoculate each one by making a 
single streak along the surface of the agar with a loopful of the suspension. 

3. Incubate the cultures for a period of two weeks. 

4. Keep careful watch of the cultures and record the extent and color of the 
growth on the various media. 

(a) What food supply is best for Azotobacter ? 

( b) Why is some soluble carbohydrate so important? 

(c) Which one of the compounds used appears to be the most 
essential for Azotobacter ? 

( d ) What are the essential elements for most bacteria? 



<0 


Exercise 21. Free Nitrogen Fixation 
Symbiotic Type 


42 


MATERIALS: 

Preserved specimens of legume nodules 
Young leguminous plant with nodules 
3 sterile Petri dishes 
3 tubes nitrogen-free agar 
3 tubes sterile water 
Sterile pipettes 
Pair of forceps 
Sterile knife 

Pure culture of Rhizobium radicicolum 

Leguminous plants, such as clovers, peas, beans, lupines, and vetches, 
when grown in moist soils bear numerous nodules or tubercles upon their 
roots. At certain times in the plant’s growth these nodules contain myriads 
of bacteria. These nodule-forming organims can fix the free nitrogen of the 
air, in which state it becomes available to the plant. Such a relation existing 
between the bacteria and the plant is one of co-operation. 

1. Remove a root from a fresh, green, leguminous plant wdth a nodule at¬ 
tached. Select as large a nodule as possible. 

2. Carefully wash the nodule in three changes of sterile water. 

3. Treat the nodule in 70 per cent alcohol for 3 to 5 minutes. Pour off the 
alcohol, and wash the nodule in three changes of sterile water. 

4. Sterilize a clean slide by heating thoroughly in the flame. 

5. When the slide is cool, place a large drop of water on it; and crush the 
nodule, with a sterile knife or scalpel in the water. 

6. Label three Petri dishes 1, 2, 3 and by means of sterile pipette, place a 
large drop of sterile water in each. 

7. By means of the sterile loop transfer one loopful of the milky material 
on the slide to the first Petri dish. Mix well. Sterilize the loop and transfer 
two loopfuls from the first Petri dish to the second. Sterilize the loop, and 
transfer two loopfuls from the second dish to the third. Mix well. 

8. Prepare a slide for staining with a loopful of the material on the sterile 
slide. 

9. Melt three tubes of legume agar, cool to 45°C. and pour the three plates. 
When the agar is solid, place the plates inverted, in moist chambers at 28° C. 
for ten days or two weeks. The nitrogen-free medium known as legume agar 
has the following composition: 


Mannit (C 6 H 8 (OH 6 )_ 15 grams 

Dibasic potassium phosphate(K 2 HP0 4 )_ 0.20 “ 

Magnesium sulphate (MgS0 4 )_ 2.20 “ 

Sodium chloride (NaCl)_ 0.20 “ 

Calcium sulphate (CaS0 4 )_ 0.10 

Calcium carbonate (CaC0 3 )_ 5.00 


Water__1000.00 

Agar_ 15.00 











43 


10. When dry, fix the preparation made from the crushed nodule, and stain 
in dilute (1:5) fuchsin for three minutes. 

11. Examine, draw and describe the plant on the desk. Leave the roots in 
the water for this purpose. Carefully examine the nodules. Note their shape, 
color, position, and size. 

12. Examine the stained preparation made from the nodule. Look very 
carefully for irregular shaped cells: pear, club, Y and T-shaped individuals. 
It is quite possible that all of these shapes may not be seen in the same prepa¬ 
ration. The bacteria when appearing in these shapes are called baderoids. 
Draw and describe the organisms. See instructor’s demonstration. 

13. Draw and describe typical nodules from the preserved specimens of red 
clover, alfalfa, soybeans, garden peas, and lupine. 

14. Prepare a sjide from one of the colonies marked by the instructor and 
examine the organisms as before. 

(a) Are there any bacteroids? 

( b ) What kind of organisms are present? 

15. Examine the pure cultures of Rhizobium radicicolum furnished. Draw 
and describe the culture according to the outline of the study of cultures. 

16. Examine the organisms after staining them with crystal violet or carbol 
fuchsin. Be sure that some of the gelatinous growth is on the slide. The 
organisms will appear unstained on a stained background. 

17. Draw and describe the organisms as seen with the oil-immersion 
objective. 


18. 


(a) What was the shape of the organisms as seen in the nodule? 
As seen from the colony? As seen from the streak culture? 

( b) What type of structure was revealed in the organisms from 
the nodules? 

(c) What are bacteroids? 

( d ) What is the difference between the organisms in the nodules 
and those in the pure cultures? 

(e) What relation do these organisms bear to the nitrogen supply 
of legumes? 

(0 Why are legumes such good crops for the land? 

(g) What is meant by inoculation of legumes? 

(/?) When is inoculation necessary? 






44 


Exercise 22. Cellulose Fermentation 


MATERIALS: 

2 20 c.m. test tubes 

100 c.c. of Omeliansky’s solution. 

A suspension of cow manure 
4 strips of filter paper 
25 c.c. graduate 
Vaseline 

Cellulose is the main constituent of the woody fiber of all plant tissue. In 
soils, marshes, the bottoms of ponds and rivers, manure piles, and compost 
heaps, cellulose is constantly undergoing decomposition. The purest forms of 
cellulose are quickly attacked by bacteria, and the progress of the decomposi¬ 
tion of a substance like filter paper is easily observed. 


Ammonium sulphate (NH 4 ) 8 S0 4 )_ 

Magnesium sulphate (MgS0 4 )_ 

Di-basic potassium phosphate (K 8 HP0 4 )_ 

Calcium carbonate (CaC0 3 )_ 

Sodium chloride (NaCl)_ 

Water_ 


0.5 grams 
0.5 “ 

1.0 “ 

5.0 “ 


a trace 
1000.0 “ 


This solution is called Omeliansky’s solution. 

1. Fill two large test tubes one-third full of the culture solution on the desk. 
See that the solution is well agitated before taking any of it, as the insoluble 
substances settle out. 

2. Add about 5 cc. of a suspension of cow manure to each tube. 

3. Immerse two strips of filter paper in a vertical position. 

4. Cover the surface of the liquid with about one-half inch of melted vase¬ 
line to exclude air. 

5. Sterilize one of the tubes to prevent bacterial growth. Observe the 
precautions of Exercise 7, when labeling the tubes. 

6. Incubate both tubes at 37° C. 

7. Compare the tubes after changes in the paper are apparent. 

8. Draw and describe the appearance of the paper. 

9. (a) What is the odor like in the tubes? 


( b ) What is the origin of the odor? 

(c) How has the color of the paper and deposits changed? 

( d) Chemically what kind of a substance is filter paper and what 
does it yield on hydrolysis? 

(e) What are the by-products of cellulose fermentation? What 
service do they render in the soil? 

(0 Why is an abundance of cellulose necessary in the heating of 
manure? 

( g) What kinds of manure heat? 

(/?) Give three additional instances where cellulose fermentation 
is important in farm practice? 









45 


Exercise 23. Hydrogen Sulphide Formation from 
Protein 

MATERIALS: 

3 tubes of egg gelatin 
3 tubes of egg agar 
Garden soil 

Cultures of: Escherichia coli 
Bacillus mycoids 
Bacillus grave ole ns 

1. Observe the precautions in handling gelatin, and prepare a stab culture 
of each of Escherichia coli , Bacillus mycoides and Bacillus gravcolens. 

2. Incubate the cultures at 20° C. 

3. Melt the agar and add about 0.1 gram of sterile lead carbonate to each 
tube. Mix well by rolling the tubes between the hands. 

4. Inoculate one tube, after cooling to 45° C. with a loopful of garden soil. 
Mix well. Inoculate the second tube with one loopful from the first. Mix 
well. Inoculate the third tube from the second, using three loopfuls. 

5. When the agar is solid incubate the culture at 30° C. 

6. After one week examine the agar shake cultures for black colonies, and 
the gelatin stab cultures for liquefaction. 

7. If the gelatin has liquefied, notice the odor and add a few drops of 5 per 
cent lead acetate solution to each tube. Observe the results? 

(a) What is the black substance formed upon the addition of 
lead salts? 

( b ) What is the source of the hydrogen sulphide? 

(c) How do you explain the power of these organisms to liquefy 
gelatin and produce “lead blackening”? 

( d) From what compound in the media is the H s S produced? 

(e) Where do these organisms occur in nature? 

(0 What is the practical value of these organisms? 





46 


Exercise 24. Sulphate Reduction 
MATERIALS: 

100 cc. of sulphate medium 
3 tubes of sulphate agar 

One two ounce bottle with cork and string 
Old sewage 

SOLUTION FOR SULPHATE REDUCTION 


Di-basic potassium phosphate (K 2 IIP0 4 )__ 0.5 grams 

Sodium lactate (NaC 3 H 5 0 3 )_ 0.5 “ 

Ammonium sulphate (NH 4 ) a S0 4 )_ 2.0 “ 

Ferrous sulphate (FeS0 4 -f 7H 2 0)_ trace 

Tap water_ 1000.0 cc. 


1. Fill the bottle to the neck with the solution and inoculate it with one cc. 
of sewage. Stopper tightly and tie in the cork. 

2. Melt the tube of sulphate agar and cool it to 45 degrees G. Inoculate it 
with a few loopfuls of sewage. Mix well and solidify quickly in cold water. 

3. Incubate the cultures at 30° C. for one week. 

4. Examine the cultures and uncork the bottle. Describe the changes. 

5. (a) What causes the change in color? 

(b) What is the odor? 

(c) Under what kind of conditions as to oxygen did the change 
take place? 

( d ) What are the chemical changes? 

( e) What other process studied was similar to this? 










■ 




. 










A 

* 


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. 

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. 

. 


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. 

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PART IV 


DAIRY BACTERIOLOGY 

Exercise 25. The Bacteriological Examination of Milk 

MATERIALS: 

11 tubes of lactose agar 
11 sterile Petri dishes 

3 99 c.c. water blanks 

4 9 c.c. water blanks 
Case sterile pipettes 
Sample of high-grade milk 
Sample of market milk 


Milk that is to be examined must be well mixed. Creaming and settling 
have some effect upon the distribution of bacteria in milk. 




FIG. 18—DIAGRAM OF THE DILUTIONS FOR ORDINARY MARKET MILK 



















48 


Samples collected for bacteriological examinations are usually taken in 
sterile, wide-mouthed, glass-stoppered bottles, packed in ice, and examined as 
soon as possible. 

Ordinary milk contains so many bacteria that it has to be diluted greatly in 
order that such a number of colonies will be present on the plates, that the 
counting may be done easily and accurately and that the results may be of 
value. 

1. Prepare lactose-agar plates from high-grade milk and ordinary market 
milk. Make duplicate dilutions according to the outline in Figs. 17 and 18. 
Be sure that all dilutions are well shaken. Make duplicate plates of the high 
grade milk with 1/1000 cc. and 1/10.000 cc. and of the market milk with 
1/1000 cc., and 1/10,000 and 1/100,000 cc. Make one blank for a control. 

2. Label each dish with the kind of milk, dilution, and the desk number be¬ 
fore making the dilutions. 

3. Melt the agar and cool it to 45 degrees C. before pouring the plates. 

4. Place the plates, inverted, in the incubator for 48 hours or one week at 
room temperature. 

5. Count the colonies, using a plate counter if necessary. When giving the 
final count per cc. of the milk keep in mind the number of colonies allowable 
on agar plates. Arrange the results in tabular form as shown in Table VI. 
Place the table on the regular note paper. 


TABLE VI.—Bacterial content of milk 


Name of 
sample 

Dilution 

Colonies 
on plate 

Bacteria 
per cc. 

Number 
of kinds 

Remarks 



I 





II 







I 





II 





I 





II 





I 





II 











6. (a) How do the total number and the kind of colonies compare in 

the two milk samples? 

(b) Does each colony represent a single organism? 

(c) Why was lactose agar used for the plates? 


























49 


( d ) How would you make plates containing 1/1,000,000 and 
1/10,000,000 cc. of milk. 

( e ) Why not use 0.1 cc. in preference to 1.0 cc. for the inoculation 
of the plates? 

(/*) What sometimes causes colonies to be in a mass in the center 
of a dish or in a mass near one side? 

( g ) Where does the moisture come from that condenses on the 
covers of the dishes? 

( h ) How does the total number of bacteria in milk compare with 
the number in soil? 

(i) Why is it not desirable to average the counts of all the plates 
in getting the final results? 

0) What are spreading colonies? In what way do they affect 
plate counts? 








W 



















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•• 



1 







r 






* 













» 







- 





«» 










t - ■ 

;■ 




# 




i 















Exercise 26. Milk Contamination 


50 


MATERIALS: 

10 tubes of lactose agar 
10 sterile Petri dishes 
1 tube of sterile milk 

3 9cc. water blanks 

4 99cc. water blanks 
Case of pipettes 
Sample of aseptic milk 
Cow hairs 

A 1 to 1000 suspension of manure 
Small pail containing skimmed milk 

PART I. 

Contamination from the Interior of the Udder, Aseptic Milk, 

The milk upon the desk was procured as follows: The cow’s udders were 
carefully cleaned. The milker’s hands were carefully washed in a disinfectant 
and the milk drawn in as nearly a dust free atmosphere as possible. The first 
streams of milk were throwm away and then the milk was drawn as carefully 
and quickly as possible into a sterile flask. In this manner the danger from 
outside contamination was reduced to the minimum. 

1. Melt two tubes of lactose agar and cool to 45° C. Dilute 1 cc. of the milk 
in a 9 cc. water blank and prepare one lactose agar plate, using 1 cc. of the 
dilution. Dilute 1 cc. of the milk in a 99 cc. water blank. Prepare a lactose 
agar plate, using 1 cc. of this dilution. Be sure that each one of the plates is 
labeled with the dilution, name of the milk, and the desk number. Incubate 
the plates, inverted, in the incubator one week. 

2. Set the sample of milk away in the locker and allows it to incubate for 
the same length of time as the plates. 

3. Observe the number of colonies that have developed on the plates. 
Tabulate the results on the regular note paper as in Table VII. 


Table VII.—Bacterial Contents of Aseptic Milk 


Dilution 

Colonies 
on plate 

Bacteria 
per cc. 

Number and 
types of kinds 










PART 2. 

Contamination from Cow Hairs 

1. Melt a tube of lactose agar. Pour the agar into a Petri dish and allow 
it to solidify. 

2. When the agar is solid, with sterile forceps carefully remove two or three 
cow hairs from the dish upon the desk and place them upon the surface of the 
agar. Make sure that the hairs are in contact with the surface of the culture 
medium for their entire length. Press them dowm with the sterile loop. Be 
careful not to break the surface of the medium. 
















51 


3. After the plate has incubated in the desk from four to six days, examine 
it. Observe the kinds of bacteria and the relative number of each. Distin¬ 
guish the molds from the bacteria. Draw the hair with the bacteria and mold 
growths. 

4. Drop a cow hair into a tube of plain sterile milk. Leave the tube in the 
incubator from four to six days. Note the kind of fermentation that takes 
place. 

PART 3 

Contamination from Manure 

1. There is a suspension of one gram fresh cow manure in 999 cc. of sterile 
water on the desk. 

2. Place 1 cc. of the 1/000 manure suspension in a 99 cc. water blank and 
place 1 cc. of this dilution in each of the two Petri dishes, using a sterile 
pipette. With another sterile pipette place 1 cc. of the 1 to 100,000 dilution in 
a 9 cc. water blank and use 1 cc. of it for each of the two plates with a dilution 
of 1 to 1,000,000. 

3. When the agar is melted, cool it to 45° C. and pour the four plates. 
Place them in the incubator for 48 hours. 

4. Observe the different kinds of colonies. Notice the relative number of 
the different kinds. 

5. Count the colonies and estimate the number of organisms per gram of 
manure. Tabulate the results on the regular note paper as in Table VIII. 


PART 4. 

Contamination from Utensils 


Dilution 

Colonies 

Per plate 


Bacteria 
per gram 

Number and 
types of kinds 

1/100,000 gm. 

I 

I 




I 

II 




1/1,000,000 gm. 

I 

I 




II 

II 





1. Pour the milk out of the pail to be found on the desk and rinse the pail 
well with tap water. Drain as dry as possible. 

2. Pour in 100 cc. of sterile water, close and shake well. 

3. Place 1 cc. of this wash water in a sterile Petri dish. 

4. Rinse the can with a cupful of boiling hot water and drain dry. 

5. Pour in 100 cc. of sterile water, close and shake well. 

6. Place one cc. of this wash water in a sterile Petri dish. 

7. Boil 500 cc. of water in the can for 5 minutes. 

8. Place 1 cc. of this water in a Petri dish. 

9. Be sure that the Petri dishes are labeled with the number of the washing 
and desk number. Use lactose agar and pour each plate. 

10. Place the inverted plates in the incubator for 48 hours. 

11. Tabulate the results on the regular note paper, showing the relation of 
bacterial content of washings to treatment. See Table IX. 














* 




52 

TABLE IX.—Reduction of Bacteria in Milk Utensils Due to Heating 


Treatments 

Bacteria per cc. 
of wash water 

Types of bacteria 

1 



2 



3 


. 


(а) Are there many kinds of colonies on the aseptic milk plates? 

(б) Did any changes take place in the milk? What was the 
nature of the change if any? 

(c) What happened in the case of the cow hair in milk? 

(d) If the moisture content of manure is 80 per cent, what is the 
bacterial content per gram of the dry manure? 

(e) How does the total number of bacteria in manure compare 
with the number in milk and soil? 

(f) If 0.56 gram of dry manure falls into each gallon of milk from 
a dirty cow, how many organisms per cc. will be introduced 
into 20 pounds of milk from the same cow? Tquart=0.946 
liter=2.08 pounds. 

(g) How can the contamination of milk from milk utensils be 
controlled? 

(h ) Which treatment was most efficient? 

(i) What construction in pails is best? 

(J) How are pails and utensils usually treated? 

(k) How is sterilization in good dairies usually accomplished? 

(/) Name the most important sources of milk contamination in 
order of their importance? 










Exercise 27. Milk Fermentation 

Acid Fermentation Caused by Streptococcus Lactis 
MATERIALS: 

a. 1 tube of plain broth 

b. 1 Durham tube of lactose broth 

C. 1 Durham tube of saccharose broth 

d. 1 Durham tube of glycerin broth 

e. 1 fermentation tube of dextrose broth 

f. 1 lactose agar slope 

g. 3 tubes of lactose agar for plates 

h. 1 tube lactose gelatin 

i. 1 small tube of plain milk 

j. 1 small tube of litmus milk 

k. 1 large tube of plain milk 

l. 4 sterile Petri dishes 

m. Ice water for gelatin 

n. Culture of Streptococcus lactis 

0. Outfit for determination of acidity 

The souring or formation of acid in milk is the most common change ob" 
served in it and may be caused by a great variety of bacteria. The souring o^ 
.milk without the formation of gas and with the production of the pleasant 
buttermilk flavor and odor is due to a group of bacteria of which Streptococ¬ 
cus lactis is the principal representative. 

1. Label each of the culture tubes and the Petri dishes with the number of 
the exercise and the letter indicating the kind of culture medium. 

2. Prepare loop dilution plates for colony formation. Label sterile Petri 
dishes number 1, 2, and 3. Melt three tubes of lactose agar and cool them not 
lower than 45° C. Label one tube number 1; one, number 2; and the remain¬ 
ing one, number 3. Transfer one loopful of the culture to the agar tube num¬ 
ber 1. Mix the organisms and agar well. Transfer 3 loopfuls of the agar and 
organisms from tube number 1 to tube number 2. Mix well. Transfer 4 
loopfuls from tube number 2 to tube number 3. Pour the agar in tube num¬ 
ber 1 into Petri dish number 1, and the agar in tube number 2 into Petri dish 
number 2, and that of tube number 3 into Petri dish number 3. This gives a 
dilution of the organisms and enough scattered colonies on one of the plates 
so that the colonies may be studied in detail. 

3. Make a stab culture in the gelatin. 

4. Inoculate all of the remaining tubes of media by means of the wire loop. 

5. Incubate the gelatin culture in the ice box. Incubate all of the other 
cultures 5 to 7 days at room temperature. 

6. Record the morphological, cultural, and biochemical properties of the 
organisms according to the outline in Appendix A. The description should be 
recorded on the regular note paper, following the order given below: 

A. Physiology 

1. Relation to Oxygen: aerobic, anaerobic, faculative. 

2. Acid production: from dextrose, lactose, saccharose, glycerin. 

3. Gas Production: from dextrose, lactose, saccharose, glycerin. 

4. Production of pigment: fluorescent, violet, blue, green, yellow, orange, 
red, brown, pink. 



54 


B. Cultural Characteristics 

1. Gelatin Stab. Draw the culture. 

(a) Growth: uniform, best at top, best at bottom. 

(b) Line of puncture: filiform, beaded, papillate, villous, arbores¬ 
cent. 


( c ) Liquefaction: none, crateriform, nap if orm, infundibuliform, 
saccate, stratiform. 

2. Agar Slope. Draw the culture. 

(a) Growth: scanty, moderate, abundant, none. 

(b) Form of growth: filiform, echinulate, beaded, spreading, 
arborescent, rhizoid. 

(c) Elevation of growth: fat, effuse, raised, convex. 

(d) Lustre: glistening, dull. 

(e ) Topography: smooth, contoured, rugose. . 

(0 Optical Characters: opaque, translucent, opalescent, iridescent. 

( g ) Chromogenesis: - photogenic, fluorescent. 

(h) Odor: absent, decided, resembling _ 

(«) Consistency: butyrous, viscid, membranous, brittle. 

(J) Medium: grayed, browned, reddened, blued, greened. 

3. Broth Culture. 

(a) Surface growth: ring, pellicle, flocculent, membranous, none. 

(b ) Clouding: slight, moderate, strong, transient, presistent, none, 
fluid, turbid. 

(c) Odor: absent, decided, resembling _ 

(d ) Sediment: compact, flocculent, granular, flaky, viscid, on 
agitation, abundant, scant, none. 

4. Agar Colonies. Draw a colony. 

(a) Growth: slow, rapid. 

( b) Form: punciiform, circular, irregular, myceloid, filamentous, 
rhizoid. 

(c) Surface: smooth, rough, concentrically ringed, radiate. 

(d) Elevation: flat, effuse, raised, convex, pulvinate, umbinate. 

(e) Edge: entile: undulate, lobate, erose, filamentous, curled. 

(0 Internal structure: amorphous, finely, coarsely-granular, 
filamentous, curled, concentric. 

5. Milk Culture. Draw the litmus milk culture. 

(a) Reaction to litmus. 

( b ) Curd: hard or soft; solid or porous. 

(c) Whey: little or abundant; clear or turbid. 

(d ) Digestion of curd: (Examine the plain milk culture). 

(e) Odor. 

(0 If curd has not formed, will if form on heating the culture 
to 100° C? Use tube I. 

(g) Percentage of acid formed. 

Note: Titrate 10 cc. of the milk from tube K with N/10 NaOH and using 
phenolphthalein as an indicator. Calculate the per cent of acid using the 
following formula: 


cc. N/10 NaOHX0.009X100 


Per cent acid = 


cc. milk used. 






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55 


C. Morphology 

1. Vegetative Cells. 

(a) Form: spheres, short rods, long rods, filaments, commas, short 
spirals, long spirals, curved. 

(b) Arrangement: single, pairs, chains, fours, cubical pockets. 

(c) Limits of size_size of majority. 

(d) Ends: rounded, truncate, concave. 

2. Capsules. 

(a) How stained_ 

3. Motility. 

4. Endospores: present, absent. 

(a) Location of endospores: central, polar. 

(b) Form: spherical, elliptical, elongated. 

5. Staining Reactions. 


6 . 


(a) 

(b) 

(a) 


( b) 

(c) 

(d) 

(e) 
(/) 


Stain a preparation from the agar slope with carbol fuchsin. 
Stain a preparation from the litmus milk culture with methy¬ 
lene blue. 

Judging from the results obtained with the different kinds of 
media, under what conditions does Streptococcus ladis grow 
best? 

What is the texture and flavor of milk soured by this organ¬ 
ism? 

Of what value is it in the dairy industry? 

Does the organism produce spores? 

Is all of the sugar of the milk fermented? If not, why not? 
What is the best temperature for the growth of the Streptococ¬ 
cus ladis group of organisms? 






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56 


Exercise 28. Milk Fermentation 

Acid Fermentation Caused by Aerobacter aerogenes group 
MATERIALS: 

a. 1 tube of plain broth 

b. 1 Durham tube of lactose broth 

C. 1 Durham tube of saccharose broth 

d. 1 Durham tube of glycerin broth 

e. 1 fermentation tube of dextrose broth 

f. 1 lactose agar slope 

g. 3 tubes lactose agar for plates 

h. 1 tube gelatin 

i. 1 small tube of plain milk 

j. 1 small tube of litmus milk 

k. 1 large tube of plain milk 

l. 4 sterile Petri dishes 

m. Ice water for gelatin 

n. Culture of Aerobacter aerogenes 

0. Outfit for determination of acidity 

A number of acid-forming bacteria also produce gas and undesirable flavors 
and odors in milk. On account of the second characteristic, their presence is 
very undesirable in milk, especially in that which is to be used for the manu¬ 
facture of butter and cheese. 

One of the organsims occuring in the intestines of cattle produces acid and 
gas from most sugars. This organism is A erobacter aerogenes. Because manure 
so frequently gets into milk, most milk under favorable temperature condi¬ 
tions will show gas formation. 

1. Label each of the culture tubes and the Petri dishes with the number of 
the exercise and the letter indicating the kind of culture medium. 

2. Prepare loop dilution plates for colony formation. Label 3 sterile Petri 
dishes 1, 2, and 3. Melt three tubes of lactose agar and cool them not lower 
than 45° C. Label 1 tube number 1, one number 2, and the remaining one 
number 3. Transfer one loopful of the culture to the agar tube number 1. 
Mix the organisms and agar well. Transfer 3 loopfuls of the agar and organ¬ 
isms from tube number 1 to tube number 2. Mix well. Transfer 4 loopfuls 
from tube number 2 to tube number 3. Pour the agar in tube number 1 into 
Petri dish number 1, and the agar in tube number 2 into Petri dish number 2, 
and that in tube number 3 into Petri dish number 3. This gives a dilution of 
the organisms and enough scattered colonies on one of the plates so that the 
colonies may be studied in detail. 

4. Make a stab culture in the gelatin by thrusting the inoculated straight 
wire into the center of the solid medium nearly to the bottom of the test tube. 
Be sure the wire is perfectly straight. 

5. Inoculate all of the remaining tubes of media by means of the wire loop. 

6. Incubate the gelatin culture in the ice box. Incubate all of the other 
cultures 48 hours in the incubator. 

7. Record the morphological, cultural, and biochemical properties of the 
organism according to the outline in Appendix A. The description should be 
recorded on the regular note paper following the order given below: 



57 


A. Physiology 

1. Relation of Oxygen; aerobic, anaerobic, faculative. 

2. Acid Production: from dextrose, lactose, saccharose, glycerin. 

3. Gas Production: from dextrose, lactose, saccharose, glycerin. 

4. Production of Pigment: fluorescent, violet, blue, green, yellow, 
orange, red, brown, pink. 

B. Cultural Characteristics 

1. Gelatin Stab. Draw the culture. 

(a) Growth: uniform, best at top, best at bottom. 

(b) Line of puncture: filiform, beaded, papillate, villous, arbore¬ 
scent. 

(c) Liquefaction: none, crateriform, napiform, infundibuliform > 
saccate, stratiform. 

2. Agar Slope. Draw the culture. 

(a) Growth: scanty, moderate, abundant, none. 

(b) Form of growth: filiform, echinulate, beaded, spreading, 
arborescent, rhizoid. 

(c) Elevation of growth: flat, effuse, raised, convex. 

(d ) Lustre: glistening, dull. 

(e) Topography: smooth, contoured, rugose. 

(0 Optical characters: opaque, translucent, opalescent, iridescent. 

( g) Chromogenesis: _photogenic, fluorescent. 

(/?) Odor: absent, decided, resembling. 

(i ) Consistency: butyrous, viscid, membranous, brittle. 

(jf) Medium: grayed, browned, reddened, blued, greened. 

3. Broth Culture. 

(a) Surface growth: ring, pellicle, flocculent, membranous, none. 

(b ) Clouding: slight, moderate, strong, transient, presistent, none, 
fluid, turbid. 

(c) Odor: absent, decided, resembling _ 

(rf) Sediment: compact, flocculent, granular, flaky, viscid on 
agitation, abundant, scant, none. 

4. Agar Colonies. Draw a colony. 

(a) Growth: slow, rapid. 

(b) Form: pundiform, circular, irregular, myceloid, filamentous, 
rhizoid. 

(c) Surface: flat, effuse, raised, convex, pulvinate, umbonate. 

(d) Edge: entire, undulate, lobate, erose, filimentous, curled. 

(e) Internal structure: amorphous, finely-, coarsely-granular, 
filamentous, curled, concentric. 

5. Milk Culture. Draw the litmus milk culture. 

(a) Reaction to litmus. 

( b ) Curd: hard or soft, solid or porous. 

(c) Whey: little or abundant; clear or turbid. 

(d) Digestion of curd: (Examine the plain milk culture). 

(e) Odor. 

(f) If the curd has not formed, will it form on heating the culture 
to 100° C? Use tube I. 

( g ) Percentage of acid formed. See Exercise 27. 




• » 











58 


C. Morphology 

1. Vegetative Cells. 

(а) Form: spheres, short rods, long rods, filaments, commas, short 
spirals, long spirals, curved. 

(б) Arrangement: single, pairs, chains, fours, cubical packets. 

(c) Limits of size-size of majority. 

(d) Ends: rounded, truncate, concave. 

2. Capsules. 

(a) How stained_ 

3. Motility. 

4. Endospores: present, absent. 

(a) Location of endospores: central, polar. 

(b) Form: spherical, elliptical, elongated. 

5. Staining reactions. 

See Exercise 27. 

6. (a) Is the organism Gram positive? 

(b) How does the per cent of acid compare with that produced by 
Streptococcus lactis.V 

(c) Does the organism produce spores? 

(d) How does the odor of the milk cultures compare with those 
fermented by Streptococcus lactis.V 

(e) What is the Wisconsin curd test? 

(0 What is its purpose? 

(g) Why are the curds kept at a high temperature? 

(h) Why is rennet added to the milk to be tested? 

(i) Do the bacteria remain in the curd or pass into the whey? 

(j) Why is dirty milk objectionable for butter and cheese making? 

( k ) In what way does the action of Aerobacter aerogenes upon 
milk differ from that of Streptococcus lactis ? 

(/) How is Aerobacter aerogenes related to the quality of milk? 

(, m ) What is the habitat of Aerobacter aerogenes ? 






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59 


Exercise 29. Milk Fermentation 

Acid Fermentation Caused by Lactobacillus Butgaricus Group 

MATERIALS: 

Raw skimmed milk 

1 125 cc. bottle, cork, and string 

Outfit for determination of acidity in milk 

There are present in ordinary milk acid-forming bacteria which develop 
only under certain conditions. If milk is kept tightly stoppered to prevent 
mold growth, and held at a comparatively high temperature, it will become 
very acid due to the growth of certain bacteria usually termed the Lacto¬ 
bacillus Bulgaricus group. 

1. Fill a 125 cc. bottle to the neck with raw skimmed milk. Cork tightly 
and secure the cork with a string as shown by the instructor. Label the 
bottle. Place in the incubator at 37° C. 

2. At the end of 2, 7, and 14 days, open the bottle and examine the milk as 
to odor, acidity (by titration), and type of organism present. Microscopic 
preparations should be made after thorough shaking of the bottle. Some of 
the curd should be spread as thinly as possible on the slide. Methylene blue 
is a good stain to use. 

3. Tabulate the observations on the regular note paper, showing the time, 
acidity, and morphology of the organisms present. See Table X. 


TABLE X.—The Development of High-Acid Organisms in Milk 


Incubation period 

Drawing of 
organisms present 

Acidity of milk 

2 days 



7 days 


♦ 

14 days 




4 . 


(a) How does the final acidity of the milk compare with that 
produced by Streptococcus lactis and Aerobacter aerogenes. 

( b ) What type of organism finally predominates? 

(c) Is the flavor of the milk desirable or undesirable? 

(d) What is the significance of the organism in fermented milk 
drinks? 















/ 


' 

■ 

' 

■' l - . . • 

. 


i ... .. . ; 

. 


















60 

(e) Who advanced a theory of human longevity connected with 
this organism? 

(0 Are the organisms of this type of great practical importance? 
Explain the answer. 

(g) What agricultural industry makes use of this organism? 



Exercise 30. Milk Preservation 


61 


MATERIALS: 

6 sterile test tubes 

Sample of raw skimmed milk 

It is very desirable to keep milk in as nearly the natural condition as possible 
until it is consumed. Bacteria are responsible for practically all of the changes 
that render milk undesirable for food purposes. With even the best methods 
of production, the contamination of milk is so great and the growth of bacteria 
so rapid that the milk becomes undesirable for food purposes in a short time. 
On this account methods for the preservation of milk are necessary. 

1. Fill each of 3 sterile tubes one-third full of sweet milk. 

2. Label the tubes and store one in the ice box, one in the desk and one in 
the incubator. Examine them at each laboratory period until the milk is 
curdled. Tabulate the time of curdling. 

3. Fill 3 sterile test tubes one-third full of sweet milk. 

4. Heat the tubes to 90° C. in water in the cup for 30 minutes. 

5. Place one of the tubes in the ice box. Place one tube in the incubator. 
Permit the other tube to remain in the desk. 

6. Examine the tubes after 24 and 48 hours. Tabulate the time of curdling 
on the regular note paper. See Table XI. 


TABLE XI.—Effect of Temperature and Pasteurization on the 

Quality of Milk 


Sample 

Treatment 

Time to curdle 

















7 . 


(a) In what order did the unheated tubes of milk curdle? 

( b) What can you say of the temperature necessary to keep milk 
sweet for as long a time as possible? 

(c) Why does it remain sweet under these conditions? 

( d ) Which tube of milk in the heated set showed signs of curdling 
or spoiling first? 





























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■ • 

• • ■■ 

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i.' 

■ > 
























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■ • 

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(e) Which tube remained “sweet” the longest? 

(/) W T hat arc the respective temperatures of the ice box, room, 
and incubator? 

(g) What can be done in storing milk in order to prolong its use¬ 
fulness? 








•v* 




























PART V. 


BACTERIOLOGY OF WATER AND SEWAGE 

Exercise 31. The Total Bacterial Content of Water 
MATERIALS: 

13 tubes of sodium caseinate agar 
13 sterile Petri dishes 
Case sterile 1 cc. pipettes 
1 sterile 10 cc. pipette 
4 9 cc. sterile water blanks 

1 99 cc. sterile water blank 

3 dextrose broth fermentation tubes 

2 samples of surface water 
1 sample of ground water 

1. Follows the dilution diagram (Fig. 19) and prepare plate cultures as 
follows: Lake water, 1/10 and 1/100 cc.; shallow well, 1 cc. and 1/10 cc.; 
ground water 1 cc. Make one “control” plate. 

2. When the duplicate plates all contain their dilutions and the sterile 
“control” is made, melt the agar and cool it to 45° C. Pour the plates, mixing 
the water and agar well. 

^ 3. When the agar is solid, incubate the plates for one week at room temper- 
ture. 



FIG. 19—DIAGRAM SHOWING DILUTIONS FOR WATER SAMPLES 


4. Use one of the water samples designated by the instructor and inoculate 
the 3 fermentation tubes after labeling as follows: 10 cc., 1 cc. and 0.1 cc. 
The 0.1 cc. is obtained by taking 1 cc. of the 1 to 9 dilution. 

5. Be careful that there are no air bubbles in the closed arm of the fermenta¬ 
tion tube and incubate the cultures 48 hours at incubator temperature. 




















64 


6. Count the colonies in the plate cultures and tabulate the results as shown 
in Table XII on the regular note paper. 

TABLE XII.—Bacterial Content of Waters 


Kind ofj 
sample ! 

Dilu¬ 

tion 

Colonies 
per plate 

Bacteria 
per cc. 

Kinds of 
colonies 

Gas from 
bacteria in 

10 cc. 

1 cc. 

0.1 cc 



























• 















7. Make a careful examination of the fermentation tubes for growth and 
gas formation. 


Note: Each fermentation tube containing gas indicates the presence of a 
member of the common intestinal bacteria. The most commonly occurring 
forms are: Escherichia coli and Aerobacter aerogencs. On the assumption that 
at least one of the organisms is present in the tube, the approximate number 
in one cubic centimeter of water may be calculated. 

(a) What is the nature of the organisms developing from ground 
water as compared to those from surface water? 

( b ) What quality of water is indicated by the results? 

(c) What chemical tests should go with these tests? 

(d) What is meant by the presumptive test for Escherichia coli? 

(e) What is the sanitary significance of Escherichia coli in water? 



































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65 


Exercise 32. The Bacteriological Examination of Sewage 
MATERIALS: 

7 tubes of sodium caseinate agar 
7 sterile Petri dishes 
Case of sterile 1 cc pipettes 
3 9 cc. sterile water blanks 
3 99 cc. sterile water blanks 
Sample raw sewage 

1. Prepare dilutions of the sewage according to the scheme in Fig. 20. Pre¬ 
pare duplicate plates with 1/100,000 and 1/1,000,000 cc. Make one control. 

2. Melt the agar and cool it to 45° C. Pour the plates, and when the agar 



FIG 20—MANNER OF MAKING SEWAGE DILUTIONS 


is solid, incubate them at room temperature for one week. 

3. Count the plates and record the data as in Table XIII. 

TABLE XIII.—Bacterial Content of Sewage 


Dilution 

Colonies 
on plate 

Bacteria 
per cc. 

Kind of colonies 

1/10,000 cc. 

I 




II 


1/100,000 cc. 

I 




II 


1/1,000,000 cc. 

I 


| 


II 



(a) How does the bacterial content of sewage compare with that 
of water? 

( b ) What kinds of organisms predominate in raw sewage? 

(c) What chemical changes take place in sewage? 





























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66 


(d) Give the most important reasons for not discharging raw 
sewage into surface waters. 

(e) Why are the same culture media used for water and sewage 
examination as for the examination of soils? 

(/) What is meant by the phrase “sewage disposal?” 

(g) What takes place in sewage during biological disposal? 



























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67 


Exercise 33. The Purification of Water 
MATERIALS: 

Sterile Berkefeld filter for faucet 

0.003 per cent of solution of chloride of lime 

1 sterile test tube 

5 sterile Petri dishes 

5 tubes of sodium caseinate agar 

Case of sterile 1 cc. pipettes 

Many cities must use water which is subject to contamination with harm¬ 
ful types of bacteria. "Such water must be rendered safe. To accomplish this, 
the water may be filtered slowly through sand filters; or by the addition of 
lime and an aluminum salt, a gelatinous precipitate can be produced in the 
water, and the bacteria will be held in the precipitate which can be removed 
by coarse filters. Chemicals which destroy bacteria and at the same time are 
not injurious to the consumer, are sometimes used to purify drinking water. 
Calcium hypochlorite is frequently used for this purpose. 

1. Collect some tap water in a sterile test tube and prepare a plate culture 
with 1 cc of the water. 

2. Add 1 cc. of a 0.003 per cent solution of chloride of lime to the 9 cc. of tap 
water in a sterile test tube and shake. This gives a solution of 1 part of chlorine 
in 1,000,000 parts of water. Permit the tube to stand 15 minutes and pre¬ 
pare two 1 cc. plate cultures from it. 

3. Collect some tap water as it passes through a sterile Berkefeld filter. 
Prepare two 1 cc. plate cultures from it. 

4. Incubate the cultures at room temperature for one week. 

5. Examine the 5 culture plates; count and record the number of bacteria 
in the water before and after treatment. 

(a) What was the effect of filtering water and of treating it with 
hypochlorite? 

( b ) Which method was the more efficient? 

(c) Which method is the more practicable for water supplies? 

( d ) What are the objections to unglazed porcelain filters? 

(e) What is the principle upon which hypochlorite is supposed to 

work in destroying bacteria? 

(/) What is the practical objection to the hypochlorite treatment 
of water supplies? 




PART VI. 


BACTERIOLOGY OF ANIMAL DISEASES 

Exercise 34. Preparation of General Disinfectant 
MATERIALS: 

A 20 per cent solution of corrosive sublimate (HgCl s ) in con- 
concentrated HC1 
2 liter stone jar 
Graduated cylinder 

As many of the pathogenic organisms produce diseases in man, as well as in 
the lower animals, an efficient disinfectant must be used in the laboratory 
when working with pathogenic organisms. 

A convenient disinfectant to use is corrosive sublimate or mercuric chloride 
(HgCl 8 ) in a 1 to 1,000 solution. 

1. Prepare 1.5 liters of corrosive sublimate, 1 to 1,000 from the 20 per cent 
solution on the desk. The 20 per cent solution is made with concentrated 
commercial hydrochloric acid (HC1) because mercuric chloride is quite insol¬ 
uble in water. 

2. Dilute the strong solution at the rate of 5 cc. to 1,000 cc. of water which 
gives approximately a 1 to 1,000 solution. Make the dilution in the stone jar 
and rinse the graduate well, pouring the rinsings into the solution. 

3. The solution is to be used on a sponge in wiping up the surface of the 
desk at the end of the laboratory period. If any material containing patho¬ 
genic organisms is accidentally spilled on the desk or floor, it should be covered 
at once with a cloth saturated with the mercuric chloride solution (1:1000). 

(a) Give the places where mercuric chloride cannot be used and 
explain why. 

(b) Is mercuric chloride a germicide or an antiseptic? 

(c) Why must gold rings and other jewelry not come in contact 
with solutions of mercuric chloride. 




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69 


Exercise 35. Bacteriology of Anthrax or Splenic Fever 

MATERIALS: 

3 plain agar slopes 

1 plain broth 

1 9 cc. water blank 

Inoculated animal for post-mortem 

Sections showing organisms 

Slides 

Eosin-methylene blue stain for blood 
Culture of Bacillus anthracis 

The discoverer of the cause of tuberculosis, Robert Koch, set down four 
laws or rules for the study and proof that any specific organism causes a specific 
disease. These are known as Koch’s postulates and are: (1) The organism 
must be demonstrated in the circulation of tissues of the diseased animals; 
(2) organism must be isolated and cultivated in artificial media outside the 
body, and successive generations of a pure culture of that organism must be 
obtained: (3) such pure cultures must, when introduced into a healthy and 
susceptible animal, produce the specific disease; (4) the organism must be re¬ 
isolated from the blood or tissues of the inoculated animal. 

It is true that there are some diseases in which the specific organism cannot 
be cultivated outside of the animal body, or for which the causal organism 
has not been discovered. Examples of such diseases are hog cholera and 
rabies. Such diseases do not fulfill postulates (2) and (3), yet by the majority 
they are provisionally accepted as caused by a specific organism. Anthrax 
is chosen as a typical pathogenic organism that fulfills all of the postulates of 
Koch. 

Note. —Remember that the inoculating needles should be flamed im¬ 
mediately after they have been used. 

1. From the material supplied, inoculate an agar slope and a tube of broth. 
Label the tubes with the name of the organism and the desk number. 

2. Leave the tube from which you have inoculated your culture on the desk. 
Wipe off the desk with the HgCl 2 solution. Wash your hands with 1/1000 
HgCl 3 solution, then with soap and water. 

3. Draw and describe the cultures in broth and on the agar slope. Follow 
the outline in descriptions. See Appendix A. 

4. Make microscopic preparations from the agar slope and from the broth, 
staining with carbol fuchsin and Gram’s stain. The slides are to be labeled 
with the student’s name, name of the organism, and kind of medium from 
which the specimen was taken, and the kind of stain used. Remember 
that you are working with a dangerous organism and be especially 
careful not to scatter any of the organisms in making the slides. 

5. Draw and describe the preparations on each slide. See that the in¬ 
structor examines and checks each one. 

6. Observe the instructor inoculate a guinea pig with the same anthrax 
culture that has been used for study. 

7. Clean up the desks as before. 











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70 


8. Post-mortem upon the guinea pig inoculated at the previous period. 
Carefully observe the method of procedure in the post-mortem examination 
of animals and the location of the various internal organs. 

9. With the sterile platinum loop inoculate an agar slope from the heart 
blood. Place it in the incubator for 48 hours. Make the culture at the post¬ 
mortem table, not at the desk. Make a similar culture from heart blood of a 
healthy pig killed by chloroform. 

10 Clean four slides very clean. Label two heart blood, one spleen, and one 
liver. 

11. At the post-mortem table make as thin smears as possible with the loop 
from the heart blood, spleen, and liver of the animal. Be sure the loop is 
sterilized after each operation and handle the slides with care. Make a slide 
from the heart blood of a healthy animal. 

12. Permit the smears to air-dry. When perfectly dry, fix the smears of 
liver and spleen by heating in the usual way. Stain the liver and spleen 
smears by Gram’s method. 

13. Fix the heart blood smear by standing it in methyl alcohol from 10 
to 15 minutes. 

14. Transfer without washing to eosin for 5 or 10 minutes. Wash and blot 
dry. 

15. Stain in aqueous methylene blue until the smear has a lavender or rose 
tint, usually about 3 to 5 minutes. Wash and dry. 

16. Examine first with the 16 m. m. objective until a good field is found. 

17. Examine with the oil-immersion objective. The red blood cells will be 
red. The nuclei of the white blood cells will be blue and their cyptoplasm will 
be pink with red granules. The anthrax bacilli will be blue and the capsules 
will appear clear as if unstained. 

18. Draw and describe a good field in the above preparation. 

19. Examine the agar slope culture made from the heart blood. Draw and 
describe it. Is there any growth on the slopes inoculated from the blood of the 
healthy animal? 

20. Prepare a slide from the above culture and stain it with carbol fuchsin. 
Draw and describe the organisms as seen under the oil-immersion objective. 

21. Examine, draw and describe the organisms as seen in stained sections of 
tissue. 


22. Leave all of the cultures in a tumbler on the desk in order that 
they may be sterilized. 


23. 


(a) On what kind of media, liquid or solid, do spores form most 
rapidly and why? 

(b ) There are many organisms in soil and in the dust of the air 
that grow like anthrax and appear very much like anthrax in 
cultures? How could you prove such a culture was not 
anthrax? 

(c) Why does anthrax produce a growth like a tuft of cotton in 
bouillon? 

( d ) Why is it so difficult to eliminate anthrax from an infected 
farm? 

( e ) What is the best method to prove positively that anthrax is 
the cause of the death of an animal? 




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71 


Exercise 36. The Bacteriology of Tuberculosis 
MATERIALS: 

Musuem specimens of tuberculous tissues 
Sputum contain Mycobacterium tuberculosis 
Fresh tuberculous tissues 
Acid alcohol 

1. Examine and describe the specimens of tuberculous tissues in the labor¬ 
atory. 

2. Make a uniform smear of some tuberculous sputum on a clean slide. Air 
dry and fix with gentle heat. 

3. Stain the smear with hot carbol fuchsin for five minutes by placing the 
slide in a tube of the stain heated by boiling water. Wash off under the tap all 
of the stain that will come off. Decolorize the preparations in acid alcohol 
(80 per cent alcohol with 3 per cent nitric acid) until the alcohol that runs from 
the slides is no longer tinged with red. Wash in tap water. Stain the prepa¬ 
rations one to three minutes with methylene blue. Wash in tap water. Dry 
and examine with the oil-immersion objective. The organisms will appear as 
very small red rods on a blue background. If the preparation is not satis¬ 
factory, repeat until a good one is obtained. 

4. Examine tuberculous animals and prepare two smears on clean slides 
from tuberculous glands, liver or spleen. Grasp the tuberculous material in a 
pair of dissecting forceps and carefully grind, crush and smear it over the 
center of the slide. Dry the smear in the air and fix it in the usual manner. 
Stain in the same way as with sputum. 

5. Draw a good field from each preparation, showing the organism and the 
surrounding tissue materials. Describe the organisms and note especially 
their size and shape. 

(a) What is meant by an “acid fast” organism? 

(b) Name some other acid-fast organisms besides the tubercle 
organism. 

(c) Where may they be found? 

( d ) What parts of the body are most often affected in the case of 
tuberculosis of the cow? 

( e ) What parts of the body may be affected? 

(/) What is meant by a calcified tubercle? 

( g ) What is meant by open tuberculosis? 

( h) What is meant by closed tuberculosis? 

(0 How may the disease be acquired by cattle? 

(j) How may it be acquired by hogs? 





72 


Exercise 37. The Agglutination Test 
MATERIALS: 

Agar slant cultures of Alcaligines abortus 

Physiological salt solution 

Sterile hypodermic syringe 

Diluted immune and normal rahhit serum 

Graduated sterile 0.1 cc. pipettes 

Case 1 ce. sterile pipettes 

Rack for small test tubes 

10 small test tubes 

Rabbits 

The agglutination test is a means of detecting one group of antibodies in the 
blood serum of animals naturally or artificially inoculated with bacteria. 
These substances are called agglutinins. The test is highly specific, and con¬ 
sists of a clumping of a suspension of the bacteria in salt solution when they 
are brought in contact with their specific antiserum. The content of the ag¬ 
glutinins in a given serum is determined by dilution of the serum with 0.85 
per cent sodium chloride solution. The highest dilution of a serum which will 
agglutinate or throw the bacteria out of suspension determines the titer of the 
serum. The test may be made macroscopically in test tubes or microscopic¬ 
ally with a hanging drop. 

1* Immunize a rabbit against Alcaligines abortus as follows: 

(a) First inoculation. Inject, intraperitioneally 0.5 cc. of a sus¬ 
pension of the bacteria washed off an agar slope with salt 
solution. 

(b) Second inoculation. Five days after the first inoculation 
inject the animal as before with 1 cc. of a suspension of the 
same culture. 

(c) The third inoculation may be made five days after the second 
using a suspension of 3 cc. of the culture. 

2. One week after the last injection the animal should be bled. A prelimin¬ 
ary test may be made with a few drops of blood from an ear vein. In case the 
blood gives a sufficiently high titer, 1 TOO or above, a larger quantity of blood 
may be drawn. 

3. With the help of the instructor bleed the animal directly from the heart. 
About 8 or 10 cc. of blood should be taken with a sterile syringe. After draw¬ 
ing the blood is stored in a sterile test tube at ice box temperature, for 48 
hours. 

4. The clot, which soon forms, should be separated from the walls of the 
tube after 24 hours with a sterile glass rod. The contracting clot will squeeze 
out the serum, which should be clear and of a light yellow color. The clear 
serum should be removed with a sterile fine capillary pipette and stored in a 
sterile test tube for use. 

5. Use fresh 4 days old cultures of Alcaligines abortus and prepare salt 
solution suspension as was done for the inoculation of the animal. 

6. Prepare a stock solution of 1:10 from both immune and normal sera. 
This may be done by diluting 0.1 cc. of serum in 1.0 cc. of sterile salt solution. 
The other dilutions can be made from this stock dilution. 


73 


7. With a sterile 1 cc. pipette measure 1 cc. of the bacterial suspension 
(antigen) into each of eight small test tubes. Arrange the tubes in two rows 
in the rack. Number the tubes from left to right in the two rows as follows: 
Front row: Al, A2, A3, and A4; backrow: Bl, B2, B3, and B4. Place the im¬ 
mune serum in the front row of tubes. 



FIG. 21—DIAGRAM FOR SERUM DILUTIONS 
The dilutions are made with the 0.1 cc. pipette. The proceedure shown for the immune serum is 
the same for the normal serum. 


8. Use a sterile 0.1 cc. pipette, and prepare the following dilutions of each 
of the sera; rinsing the pipette in salt solution after each dilution. Mix the 
antigen and sera after each dilution by inversion over the index finger. Wipe 
off the tip of the finger with a clean cloth after each mixing. Transfers and 
dilutions, made according to the diagram (Fig. 21) will give in tube 1,1:50; 
tube 2, 1 TOO; tube 3, 1:200; and tube 4, 1:500. 

9. After the dilutions of the sera are made carefully wash the 0.1 cc. pipette 
in salt solution and then in distilled water. 


10. Place the rack holding the tubes in the 37° C. incubator for 48 hours. 

11. Remove the rack and tubes from the incubator. Examine the tubes by 
transmitted light. All tubes in which the bacteria have not settled out have 
not agglutinated. Clumps of bacteria floating in the solution are partly ag¬ 
glutinated. 

12. Compare the dilutions made with the antiserum and with normal serum 
Tabulate the results, by dilutions for the two sera. In the diagnosis of bovine 
contagious abortion agglutinations in dilutions of 1:50 and above indicate a 
positive test. 

13. (a) What is the name of the antibodies used in this exercise? 

( b ) Name two other kinds of antibodies? 

(c) What important human disease may be diagnosed by means 
of this test? 

(d) What name is applied to it when used for the above human 
disease? 

(e) What is the value of such a test as this? 

(/) What substances in the bacteria probably stimulate antibody 
production? 

(g) How may this be related to the specificity of the serological 
tests? 

( h ) Of what value is such a test as this in the classification of 
bacteria? 


















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74 


Exercise 38. Test of Disinfectants 
MATERIALS: 

A 10 per cent solution of carbolic acid 
A 1:500 solution of mercuric chloride 
8 tubes of plain broth reaction pH 7.0. 

1 tube of sterile distilled water 

2 empty sterile test tubes 

1 box of sterile 1 cc. pipettes 
Burettes for measuring disinfectants 
Culture of Escherichia coli 
Culture of Bacillus anthracis 

Much misinformation is prevalent concerning the value of disinfectants. In 
order to gain some first-hand knowledge concerning some of the disinfectants 
in common use, two will be examined. 

Each student will test one disinfectant. The disinfectants will be assigned 
by the instructor. 

Escherichia coli will be used for example of a non-spore-forming organism 
and Bacillus anthracis will be used as an example of a sporogenous type. 

1. Make an agar streak culture of Escherichia coli and of Bacillus anthracis. 
Place the cultures in the 30° C. incubator for 48 hours. 

2. By means of a sterile 1 cc. pipette measure 4 cc. of sterile distilled water 
into each of the two sterile test tubes. Label one tube Escherichia coli and the 
other B. anthracis. 

3. By means of the sterile loop prepare suspensions of the two cultures, 
transferring organisms from the agar streak cultures. 

4. Using care to avoid contamination measure with a burette, 4 cc. of the 
disinfectant assigned, into each of the suspensions of bacteria. The dilution 
produced will allow a 5 per cent solution of phenol or a 1:1000 solution of 
mercuric chloride to act upon the bacteria. 


TABLE XIV.—Exposure Table for Disinfectant Tests 


Escherichia coli 

Bacillus Anthracis 

3 minutes 

lM hours 

10 minutes 

24 hours 

30 minutes 

48 hours 

1 Y* hours 

72 hours 











5. At the end of the periods given in Table XIV transfer a loopful of the 
suspension of each culture to a tube of sterile broth. Incubate the cultures 
48 hours at 37° C. 


6. Examine the tubes for growth, comparing them with sterile tubes if 
necessary. 

7. Construct a table on the regular note paper showing the results of the 
tests of both disinfectants, Table XV. Use a plus (+) sign when the organism 
is killed and a minus (-) sign when it is not killed. 


TABLE XV.—Test of Disinfectants 


Disinfectant 

Escherichia coli 

Bacillus Anthracis 

Exposure time in minutes 

Exposure time in hours 

3 

10 

30 

90 

1.5 

24 

48 

72 













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What is meant by sepsis? 

What is meant by antisepsis? 

What is meant by antiseptic? 

What is a disinfectant? 

What is a germicide? 

Are all antiseptics disinfectants? 

Name two gaseous disinfectants. 

What are the advantages of gaseous disinfectants? 

Give the advantages and disadvantages of the above disin¬ 
fectants for stable disinfection. 

Give the best method of procedure in disinfecting a stable. 
What is meant by “terminal” disinfection? “concurrent” 
disinfection? 


8. (a) 

(b) 

(c) 

(d) 

(e) 
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APPENDIX A 


OUTLINE FOR STUDY OF BACTERIAL CHARACTERS 


A. PHYSIOLOGY (biochemical properties) 


1. Relation to oxygen 

Notice the distribution of the growth in the fermentation tubes and 
in tubes of broth and gelatin. 

2. Pigment production. 

Notice the growth on solid media. 

3. Test reaction of all sugar broths, glycerin and plain broth by remov¬ 
ing a drop with a sterile loop and placing it on litmus paper. 

4. Note gas formation in any sugar broths and in glycerin broth. If 
gas is formed in fermentation tubes, measure it and determine the 
H:C0 2 ratio as showrn by the instructor. 

5. Observe if nitrates are reduced and if gas is formed (See Exercise 17.) 

6. Test the starch agar culture for diastasic action if there is growth. 

7. Habitat. Where the organism is found. 



FIG. 22—NONLIQUEFYING GELATIN STAB CULTURES 
1 , filiform; 2, beaded; 3, echinulate; 4, villous; 5, dborescent 








































77 


B. CULTURAL CHARACTERS 

1. Gelatin Stab. Draw the culture. (See Figs. 22 and 23.) 

I. Nonliquefying. 

(a) Line of puncture: filiform, uniform needle-shaped growth; 
beaded, succession of small, disjointed colonies; echinulate, 
Pricky; villous, beset with unbranched hair-like extensions; 
arborescent, beset with root-like extensions. 

(b) Surface growth. Same as for colonies on plate cultures. 

II. Liquefying 

(a) Type of liquefaction: crater iform, saucer-shaped; napiform, 
turnip-shaped; infundibuliform, funnel-shaped; saccate, sack¬ 
shaped; stratiform, the liquefaction descending in a horizontal 
plane. 

(b ) Character of the fluid: clear, cloudy, flocculent, granular. 

2. Streak cultures (agar or potato). Draw the agar culture. 


(a) Growth: invisible, scanty, moderate, abundant. 

( b ) Form: filiform, a narrow line; echinulate, growth along line of 
inoculation with toothed or pointed margins; beaded, a suc¬ 
cession of small, disjointed colonies; effused, spreading; 
villous, plumose, arborescent. See Fig. 24.) 

(c) Luster: glistening, dull, cretaceous. 

(d) Optical characters: opaque, translucent, opalescent, iridescent. 

(e) Elevation of growth: same as for plate cultures. 

(/) Topography. 



FIG. 24—AGAR STREAK CULTURES 
1, filiform; 2, echinulate; 3. beaded; 4. effused; 5. villous 



(g) Consistency: slimy, butyrous, of a consistency like butter; 
viscid, growth follows the needle when touched and with¬ 
drawn; coriaceous, growth tough, leathery; brittle, growth dry, 
friable under the needle. 

( h ) Medium discolored. 

3. Beef-broth Cultures 

(a) Growth or no growth. 

( b ) Condition of fluid: clear, cloudy. 

(c) Surface membrane: when formed, color, consistency. 

(d) Sediment: amount, compact, flocculent, granular, viscid. 




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78 


4. Milk Cultures. Draw the litmus milk culture. 

I. Curd formed. 

(a) Time required to curdle. 

(b) Character of curd: hard or soft, solid or porous, changed or not, 
when boiled. 

(c) Whey: amount, clear or turbid. 

(d) Reaction to litmus. 

(c) Digestion of curd (examine plain milk culture): time required, 
reaction to litmus, solution cloudy or clear. 

(/) Gas bubbles. 

(ff) Odor. 

II. Digestion without formation of curd. 

III. No visible change even after boiling. 

5. Plate Cultures. 


I. Surface colonies. Naked eye appearance. (See Fig. 25.) Draw a 
colony. 



FIG. 25—NAKED EYE APPEARANCE OF COLONIES 
A, punctiform; B, circular; C, oval; D, spindle-shaped; E, conglomerate; F, ameboid; G, rhizoid; 
H, curled; I, myceloid; J, filamentous. 


(a) punctiform, too small to be defined by the naked eye; circu¬ 
lar: oval; spindle-shaped; conglomerate; an aggregate of similar 




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79 


colonies; ameboid, very irregular; rhizoid, branched, root¬ 
like structure; curled, filaments in strands like curly hair; 
myceloid, filamentous, with the character of a mold. 

( b) Size, approximate, expressed in millimeters. 



FIG. 26—SURFACE ELEVATION OF COLONIES 
a, flat; b, spreading; c, raised ; d, convex; e, capitate; f, umbilicate; g, umbonate 

(c) Surface elevation (See Fig. 26): flat; spreading; raised; con¬ 
vex; surface a segment of a circle; capitate;surface a hemisphere 
umbilicate, depressed in the center; umbonate, elevated at 
the center. 

(rf) Topography of surface: smooth; contoured, smoothly undulat¬ 
ing, like the surface of a relief map; rugose, short, irregular 
folds due to shrinkage; verrucose, growth wartlike, with wart¬ 
like prominence. 

II. Microscopic appearance. Draw a section and edge of the colony. 



FIG. 27—INTERNAL STRUCTURE OF COLONIES 
1, amorphous; 2, finely granular; 3. coarsely granular; 4, grumose; 5, gyrose; 6, reticulate; 

7, filamentous 

1, amorphous, 2, finely granular; 3, coarsely granular; 4, grumose; 5, gyrose; 
6, reticulate; 7, filamentous 

(a) Internal structure (See Fig. 27) amorphous, no definite struc¬ 
ture; finely granular, coarsely granular; grumose, appears clot¬ 
ted; gyrose , showing chinks or cracks; reticulate, netted; 
filamentous , tangled wavy threads. 



FIG. 28—EDGES OF COLONIES 

8, entire 9, undulate; 10, repand; 11, anriculale; 12, lacerate; 13, fimbricale; 14, ciliate 
































80 


8, entire, 9, undulate; 10, repand; 11, auriculate; 12, lacerate; 13, j\imbricate 

14, ciliate 

(b) Edge of colony (See Fig. 28): entire; undulate, wavy; repand; 
auriculate; lacerate, irregularly cleft ;/imbricate, fringed; ciliate, 
finely toothed. 

III. Deep colonies. Draw a colony. 

(a) Color. 

( b ) Shape: punctiform, lanceolate, oval, circular, spindle-shaped, 
irregular, branched, filamentous. 

(c) Translucency. 

C. MORPHOLOGY 

1. Form and arrangement (examine stained organisms on slides); coccus, 
single, and grouped; diplococcus; streptococcus; rods, single and in chains; 
spirals. (See Fig. 5). 

2. Size. Measurements in terms of the micron. 

3. Reaction to stains: 

(a) Simple stains; stains easily or with difficulty. 

( b ) Differential stains: Gram’s stain. 

4. Spores: Time required for formation, medium, position in cell. 

5. Special characters. 

(a) Flagella. (Motility in hanging drop.) 

( b ) Capsule. 

(c) Granules. 

(d) Involution forms. 





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APPENDIX B 


GLOSSARY OF TERMS 

Agar-hanging block, a small block of nutrient agar cut from the poured’ 
plate and placed on a cover glass, the surface next the glass having first been 
touched with a loop from a young fluid culture or with a dilution from the 
same. It is examined upside down, the same as a hanging drop. 

Ameboid, assuming various shapes, like an ameba. 

Amorphous, without visible differentiation in structure. 

Aborescent, a branched, tree-like growth. 

Beaded, in stab, or stroke, disjointed or semi-confluent colonies along the 
line of inoculation. 

Brief, a few days, a week. 

Brittle, growth dry, friable under the platinum needle. 

Bullate, growth rising in convex prominences, like a blistered surface. 

Butyrous, growth of a butter-like consistency. 

Chains, short chains, composed of 2 to 8 elements; long chains, composed 
of more than 8 elements. 

Ciliate, having fine, hair-like extensions, like cilia. 

Cloudy, said of fluid cultures which do not contain pseudozoogloeae. 

Coagulation, the separation of casein from whey in milk. This may take 
place quickly or slowly, and as a result of either the formation of an acid or 
of a rennet-like ferment. 

Contoured, an irregular, smoothly undulating surface, like that of a relief 
map. 

Convex, surface the segment of a circle, but flattened. 

Coprophil, dung bacteria. 

Coriaceous, growth tough, leathery, not yielding to the platinum needle. 

Crateriform, round, depressed, due to the liquefaction of the medium. 

Cretaceous, growth opaque and white, chalky. 

Curled, composed of parallel chains in wavy strands, as in anthrax colonies. 

Diastasic action, conversion of starch into water soluble substances by 
diastase. 

Echinulate, in agar stroke, a growth along line of inoculation, with toothed 
or pointed margins; in stab cultures, growth beset with pointed outgrowths. 

Effuse, growth thin, veily, unusually spreading. 

Entire, smooth, having a margin destitute of teeth or notches. 

Erose, border irregularly toothed. 

Filamentous, growth composed of long, irregularly-placed or interwoven 
filaments. 

Filiform, in stroke or stab cultures a uniform growth along line of inocula¬ 
tion. 

Fimbriate, border fringed with slender processes, larger than filaments. 

Floccose, growth composed of short curved chains, variously oriented. 

Flocculent, said of fluids which contain pseudozoogleae, that is, small 
adherent masses of bacteria of various shapes and floating in the culture fluid. 




82 


Fluorescent, having one color by transmitted light and another by re¬ 
flected light. 

Gram’s stain, a method of differential bleaching after crystal violet, 
methyl violet, etc. The+mark is to be given only when the bacteria are deep 
blue or remain blue after counterstaining with a counterstain 

Grumose, clotted. 

Infundibuliform, form of a funnel or inverted cone. 

Iridescent, like mother of pearl. The effect of very thin films. 

Lacerate, having the margin cut into irregular segments, as if torn. 

Lobate, border deeply undulate, producing lobes (see Undulate). 

Long, many weeks or months. 

Maximum temperature, temperature above which growth does not take 
place. 

Medium, several weeks. 

Membranous, growth thin, coherent, like membrane. 

Minimum temperature, temperature below which growth does not take 
place. 

Myceloid, colonies having the radiately filamentous’appearance of mold 
colonies. 

Napiform, liquefaction with the form of a turnip. 

Nitrogen requirments, the necessary nitrogenous food. This is deter¬ 
mined by adding to nitrogen-free media the nitrogen compound to be 
tested. 

Opalescent, resembling the color of an opal. 

Optimum temperature, temperature at which growth is most rapid. 

Pellicle, in fluid, bacterial growth either forming a continuous or an in¬ 
terrupted sheet over the fluid. 

Peptonized, said of curds dissolved by trypsin. 

Persistent, many weeks or months. 

Plumose, a fleecy or feathery growth. 

Pseudozoogloeae, clumps of bacteria, not separating readily in water, 
arising from imperfect separation or more or less fusion of the components, but 
not having the degree of compactness and gelatinization seen in zoolgoeae. 

Pulvinate, in the form of a cushion, decidely convex. 

Punctiform, very minute colonies, at the limit of natural vision. 

Raised, growth thick, with abrupt or terraced edges. 

Rapid, developing in twenty-four to forty-eight hours. 

Repand, wrinkled. 

Rhizoid, growth of an irregular branched, or root-like character, as in 
B. mycoides. 

Ring, (same as rim), growth at the upper margin of a liquid culture, ad¬ 
hering more or less closely to the glass. 

Saccate, liquefaction the shape of an elongated sack, tubular, cylindrical. 

Scum, floating islands of bacteria, an interrupted pellicle or, bacterial 
membrane. 

Short, applied to time, a few days, a week. 

Slow, requiring five or six days or more for development. 

Sporangia, cells containing endospores. 

Spreading, growth extending much beyond the line of inoculation; that is, 
several millimeters or more. 




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Stratiform, liquefying to the walls of the tube at the top and then pro¬ 
ceeding downward horizontally. 

Thermal death point, the degree of heat required to kill young fluid 
cultures of an orgamism exposed for ten minutes (in thin-walled test tubes 
of a diameter not exceeding 20 mm.) in the thermal water bath. The water 
must be kept agitated, so that the temperature shall be uniform during the 
exposure. 

Transient, a few days. 

Turbid, cloudy, with flocculent particles; cloudy plus flocculence. 

Umbonate, having a button-like raised center. 

Undulate, border wavy, with shallow sinuses. 

Vermiform contoured, growth like a mass of worms or intestinal coils. 

Verrucose, growth wartlike, with wartlike prominences. 

Villous, growth beset with hairlike extensions. 

Viscid, growth follows the needle when touched and withdrawn: sediment 
on shaking, rises as a coherent swirl. 

Zoogloeae, firm gelatinous masses of bacteria, one of the most typical 
examples of which is the Streptococcus mesenteroides of sugar vats (Leu- 
conostoc mesenteroides), the bacterial chains being surrounded by an enor¬ 
mously thickened firm covering, inside of which there may be one or many 
groups of the bacteria. 





























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